Lipid vesicle compositions with penetration enhancing agents

ABSTRACT

The present application is related to a pharmaceutical composition a biphasic lipid vesicle comprising a lipid bilayer comprising vesicle forming lipids; an oil-in-water emulsion stabilized by one or more surfactants; one or more compounds; and one or more penetration enhancing agents. The one or more penetration enhancing agents include one or more non-ionic surfactants having a hydrophilic-lipophilic balance (HLB) of about 10 or less, alone or combination with one or more penetration enhancing agents selected from one or more of terpenes, alkaloids, salicylate derivatives, and polycationic surfactants and combinations thereof. The present application is also related to a pharmaceutical composition comprising a biphasic lipid vesicle comprising a lipid bilayer comprising vesicle forming lipids; an oil-in-water emulsion stabilized by one or more polycationic surfactants; and one or more compounds.

RELATED APPLICATIONS

The present application claims the benefit of priority of co-pendingU.S. provisional patent application No. 62/904,606 filed on Sep. 23,2019, and U.S. provisional patent application No. 62/904,584 filed onSep. 23, 2019, the contents of both of which are incorporated herein byreference in their entirety.

FIELD

The present technology generally relates to lipid vesicle formulationsfor the topical delivery of a therapeutic compound where the lipidvesicle formulation comprises one or more penetration enhancing agentssuch as one or more surfactants having an HLB of 10 or less.

BACKGROUND

The barrier properties of the skin prevent most external substances topermeate into the body. The properties of most drugs fall outside theoptimum range of permeability and hence require some type of an enhancerto be therapeutically useful. The main barrier controlling dermalprotein delivery is the outermost layer of the skin, the stratum corneum(SC). In mammalian skin, the SC (10 to 20 μm thick) consists of deadcorneocytes that are composed of cross-linked keratin and intercellularlipids organized in bilayers. Underneath the SC is the viable epidermis(50 to 100 μm) and deeper is the dermis (1-2 mm) that contains a richcapillary bed for drug absorption just below the dermal-epidermaljunction. The generally accepted size limit of molecules for passivedelivery through the skin is below 500 Da. Unassisted penetration ofmolecules above this molecular weight through intact skin is extremelylow.

Different delivery approaches have been developed to facilitate thediffusion of drugs into or through the skin. The enhanced permeationthrough the skin could be achieved by physical methods (e.g.microneedles, thermal ablation), electrical methods (e.g.electroporation, iontophoresis) or chemical methods (e.g. chemicalenhancers). Although the use of physical and electrical methods toenhance the drug permeation through the skin has shown some success inenhancing the delivery of both small and large molecules, there arestill significant hurdles to overcome before approval. Severalnon-invasive delivery vehicles, mostly lipid-based, have been developedfor protein delivery, such as, liposomes, transfersomes, niosomes andsolid lipid nanoparticles. However, these delivery systems were onlyable to deliver limited amount of proteins into the different skinlayers, as compared to the other invasive techniques.

U.S. Pat. Nos. 5,853,755 and 5,993,851 describe biphasic lipid vesiclecompositions and methods of their preparation. U.S. Pat. No. 5,993,852describes biphasic lipid vesicle compositions for transdermaladministration of an immunogen.

SUMMARY

The present disclosure includes a biphasic lipid vesicle compositioncomprising:

-   -   a) lipid vesicles each comprising a lipid bilayer comprising        vesicle forming lipids,    -   b) an oil-in-water emulsion entrapped in the biphasic lipid        vesicles, and stabilized by one or more surfactants;    -   c) one or more compounds entrapped in the lipid bilayer and/or        the oil-in-water emulsion;    -   d) one or more penetration enhancing agents entrapped in the        lipid bilayer and/or the oil-in-water emulsion;

wherein the one or more penetration enhancing agents are one or morenon-ionic surfactants having a hydrophilic-lipophilic balance (HLB) ofabout 10 or less.

The present application also includes a biphasic lipid vesiclecomposition comprising:

-   -   a) lipid vesicles comprising a lipid bilayer comprising vesicle        forming lipids,    -   b) an oil-in-water emulsion entrapped in the biphasic lipid        vesicles, and comprising one or more polycationic surfactants;        and    -   c) one or more compounds entrapped in the lipid bilayer and/or        the oil-in-water emulsion.

The present application also further includes method of preparingbiphasic lipid vesicles of the disclosure comprising:

-   -   a) preparing an oil-in-water emulsion comprising one or more        surfactants, by mixing oil components of the oil-in-water        emulsion with aqueous components of the oil-in-water emulsion,        wherein the oil components and/or the aqueous components of the        oil-in-water emulsion comprises the one or more surfactants;    -   b) solubilizing vesicle forming lipids in an acceptable solvent        other than water,    -   c) adding one or more compounds and one or more penetration        enhancing agents to the oil components and/or the aqueous        components of step a), and/or the solubilized vesicle forming        lipids of step b);    -   d) adding the oil-in-water emulsion to the solubilized vesicle        forming lipids; and    -   e) mixing the oil-in-water emulsion and the solubilized vesicle        forming lipids under mixing conditions effective to form the        biphasic lipid vesicles comprising a lipid bilayer comprising        vesicle forming lipids, and an oil-in-water emulsion entrapped        in the biphasic lipid vesicles.

The present application also further includes a method of delivering oneor more compounds by administering biphasic lipid vesicle compositionsof the disclosure topically to the skin or mucosal membrane to asubject.

The present application also includes a method of improving topicaldelivery of one or more compounds comprising administering an effectiveamount of biphasic lipid vesicle compositions of the disclosure to theskin or mucosal membrane of a subject in need thereof.

The present application also further includes a method of treating orpreventing skin conditions related to excessive or defective collagenproduction in a subject comprising administering to the subject in needthereof, an effective amount of lipid vesicle cosmetic compositions ofthe disclosure to a subject in need thereof.

The present application also further includes method of treatingdisease, disorder or condition treatable by delivering one or moretherapeutic compounds by administering a therapeutically effectiveamount of biphasic lipid vesicle pharmaceutical compositions of thedisclosure topically to the skin or mucosal membrane to a subject inneed thereof.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the disclosure, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A and B show confocal microscopic images of human skin treatedwith FIG. 1A) showing exemplary peptide lipid vesicle formulations 1-4containing a rhodamine red labelled 12mer peptide (molecular weight ofpeptide about 1200), FITC-insulin (molecular weight of insulin about6,000) and FITC-IgG (molecular weight of IgG about 150,000); and FIG.1B) showing a separate control study with Alexa 647 labelled IgG (redfluorescence) incorporated into biphasic vesicles (comparative formula);the skin sections showed minimal fluorescence throughout the epidermisand dermis in the red channel, ie. first panel (the three panels: firstpanel: red channel for Alexa IgG; second panel: general tissue stain(blue nuclear stain Syto 60); third panel: merged image); last panel:placebo formulation treated skin (red channel and general tissue stainmerged image) showing no fluorescence background at the settings usedfor analysis of protein delivery.

FIG. 2 shows confocal microscopic images of mouse skin treated withformulations nucleic acid lipid vesicle formulations F-TOM-1-5. For eachformulation three panels are shown: the first panel: red channel for RFPexpression (seen as light colored areas in the epidermis and dermis);second panel: general tissue stain (blue nuclear stain Syto 60); thirdpanel: merged image).

DETAILED DESCRIPTION

I. Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present application herein described for which theyare suitable as would be understood by a person skilled in the art.

The embodiments, illustratively described herein may suitably bepracticed in the absence of any element or elements, limitation orlimitations, not specifically disclosed herein. Thus, for example, theterms “comprising,” “including,” “containing,” etc. shall be readexpansively and without limitation. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the claimed technology.Additionally, the phrase “consisting essentially of” will be understoodto include those elements specifically recited and those additionalelements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of”excludes any element not specified.

For example, as used in this application and claim(s), the words“comprising” (and any form of comprising, such as “comprise” and“comprises”), “having” (and any form of having, such as “have” and“has”), “including” (and any form of including, such as “include” and“includes”) or “containing” (and any form of containing, such as“contain” and “contains”), are inclusive or open-ended and do notexclude additional, unrecited elements or process steps.

The term “consisting” and its derivatives as used herein are intended tobe closed terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, and also excludethe presence of other unstated features, elements, components, groups,integers and/or steps.

The phrase “consisting essentially of” will be understood to includethose elements specifically recited and those additional elements thatdo not materially affect the basic and novel characteristics of theclaimed technology. The phrase “consisting of” excludes any element notspecified

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present. The term “and/or” with respect to enantiomers,prodrugs, salts and/or solvates thereof means that the compounds of thedisclosure exist as individual enantiomers, prodrugs, salts andhydrates, as well as a combination of, for example, a salt of a solvateof a compound of the disclosure.

In embodiments comprising an “additional” or “second” component oreffect, such as an additional or second compound, the second compound asused herein is different from the other compounds or first compound. A“third” compound is different from the other, first, and secondcompounds, and further enumerated or “additional” compounds aresimilarly different.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent depending upon the context inwhich it is used. If there are uses of the term which are not clear topersons of ordinary skill in the art, given the context in which it isused, “about” will mean up to plus or minus 10% of the particular term.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the elements (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or illustrative language (e.g., “such as”) provided herein, isintended merely to better illuminate the embodiments and does not pose alimitation on the scope of the claims unless otherwise stated. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential.

The term “hydrophilic” as used herein refers to a compound or additivethat is substantially water soluble, water dispersible, or generallycapable of absorbing and/or transmitting water.

The term “hydrophobic” as used refers to a compound or additive that issubstantially non-soluble or dispersible in water.

The terms “nucleic acid” or “oligonucleotide”, as used herein means twoor more covalently linked nucleotides. Unless the context clearlyindicates otherwise, the term generally includes, but is not limited to,deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which may besingle-stranded (ss) or double stranded (ds). For example, the nucleicacid molecules or polynucleotides of the disclosure can be composed ofsingle- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis a mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typicallydouble-stranded or a mixture of single- and double-stranded regions. Inaddition, the nucleic acid molecules can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. The term“oligonucleotide” as used herein generally refers to nucleic acids up to200 base pairs in length and may be single-stranded or double-stranded.The sequences provided herein may be DNA sequences or RNA sequences orhybrid sequences, however it is to be understood that the providedsequences encompass both DNA and RNA, as well as the complementary RNAand DNA sequences, unless the context clearly indicates otherwise. Forexample, the sequence 5′-GAATCC-3′, is understood to include5′-GAAUCC-3′, 5′-GGATTC-3′, and 5′GGAUUC-3′. The nucleic acid oroligonucleotide may include naturally occurring bases including adenine,guanine, cytosine, thymidine and uracil. The sequences may also containmodified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymidine and uracil; and xanthine andhypoxanthine as well as others. The term “isolated nucleic acidsequences” as used herein refers to a nucleic acid substantially free ofcellular material or culture medium when produced by recombinant DNAtechniques, or chemical precursors, or other chemicals when chemicallysynthesized. An isolated nucleic acid is also substantially free ofsequences which naturally flank the nucleic acid (i.e. sequences locatedat the 5′ and 3′ ends of the nucleic acid) from which the nucleic acidis derived. The nucleic acid can for example be plasmid DNA, a viralvector, naked DNA, RNA, DNA/RNA hybrids and synthetic nucleic acids andthe like.

As used herein, the terms “peptide,” “polypeptide,” and “protein” referto any chain of two or more natural or unnatural amino acid residues,regardless of post-translational modifications (e.g., glycosylation orphosphorylation). The polypeptides incorporated into the biphasicvesicles of the disclosure can include for example from 3 to 3500natural or unnatural amino acid residues. Included are proteins that area single polypeptide chain and multisubunit proteins (e.g. composed of 2or more polypeptides).

The term “amino acid” includes all of the naturally occurring aminoacids as well as modified L-amino acids. The atoms of the amino acid canfor example include different isotopes. For example, the amino acids cancomprise deuterium substituted for hydrogen, nitrogen-15 substituted fornitrogen-14, and carbon-13 substituted for carbon-12 and other similarchanges.

An “immunogen” as used herein means a substance which when administeredto a subject provokes an immune response and causes production of anantibody, activate lymphocytes or other reactive immune cells directedagainst an antigenic portion of the immunogen

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, single chain, humanized and otherchimeric antibodies as well as binding fragments thereof. The antibodymay be from recombinant sources and/or produced in transgenic animals.Also included are human antibodies that can be produced through usingbiochemical techniques or isolated from a library. Humanized or chimericantibody may include sequences from one or more than one isotype orclass.

The term “binding fragment” as used herein to a part or portion of anantibody or antibody chain comprising fewer amino acid residues than anintact or complete antibody or antibody chain and which binds theantigen or competes with intact antibody. Exemplary binding fragmentsinclude without limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv,dimers, nanobodies, minibodies, diabodies, and multimers thereof.Fragments can be obtained via chemical or enzymatic treatment of anintact or complete antibody or antibody chain. Fragments can also beobtained by recombinant means. For example, F(ab′)2 fragments can begenerated by treating the antibody with pepsin. The resulting F(ab′)2fragment can be treated to reduce disulfide bridges to produce Fab′fragments. Papain digestion can lead to the formation of Fab fragments.Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies,diabodies, bispecific antibody fragments and other fragments can also beconstructed by recombinant expression techniques.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects are defined in more detail. Each aspect so defined may becombined with any other aspect or aspects unless clearly indicated tothe contrary. In particular, any feature indicated as being preferred oradvantageous may be combined with any other feature or featuresindicated as being preferred or advantageous.

The term “composition(s) of the disclosure” as used herein refers to acomposition comprising biphasic lipid vesicles described herein.

The term “penetration enhancing agents” as used herein refers to one ormore non-ionic surfactants having a hydrophilic-lipophilic balance (HLB)of about 10 or less or polycationic surfactants. In an embodiment, theone or more penetration enhancing agents are one or more non-ionicsurfactants having a HLB of about 10 or less in combination with one ormore penetration enhancing agents selected from one or more terpenes,alkaloids, salicylate derivatives, and polycationic surfactants andcombinations thereof.

The term “entrapped” as used herein refers to the non-covalentassociation of the referred-to agent with a biphasic lipid vesicle'slipid bilayer or bilayers, the biphasic lipid vesicle's central core,and/or a space or spaces between adjacent bilayers of the biphasic lipidvesicle.

The term “biphasic lipid vesicle” as used herein refers to a vesiclewhose central core compartment is occupied by an oil-in-water emulsioncomposed of an aqueous continuous phase and a dispersed hydrophobic,hydrophilic or oil phase. In an embodiment, the spaces between adjacentbilayers of the biphasic lipid vesicle may also be occupied by theemulsion.

The term “emulsion” as used herein refers to a mixture of two immisciblesubstances.

The term “bilayer” as used herein refers to a structure composed ofamphiphilic lipid molecules arranged in two molecular layers, with thehydrophobic tails on the interior and the polar head groups on theexterior surfaces.

The term “topical administration” or “topical delivery” as used hereinmeans intradermal, transdermal and/or transmucosal delivery of acompound by administration of a composition comprising the compound orcompounds to skin and/or a mucosal membrane.

The term “gemini surfactant” as used herein refers to a surfactantmolecule which contains more than one hydrophobic tail, and eachhydrophobic tail having a hydrophilic head wherein he hydrophobic tailsor hydrophilic heads are linked together by a spacer moiety. Thehydrophobic tails can be identical or differ. Likewise, the hydrophilicheads can be identical or differ. the hydrophilic heads may be anionic,cationic, or neutral.

The term “HLB” or “Hydrophilic-Lipophilic Balance” value refers tostandard HLB according to Griffin, J. Soc. Cosm. Chem., vol. 5, 249(1954), which indicates the degrees of hydrophilicity and lipophilicityof a surfactant.

The term “subject” as used herein includes all members of the animalkingdom including mammals, and suitably refers to humans. Thus themethods and uses of the present application are applicable to both humantherapy and cosmetic applications and veterinary applications.

The term “treating” or “treatment” as used herein and as is wellunderstood in the art, means an approach for obtaining beneficial ordesired results, including clinical results. Beneficial or desiredclinical results include, but are not limited to alleviation oramelioration of one or more symptoms or conditions, diminishment ofextent of disease, stabilized (i.e. not worsening) state of disease,preventing spread of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, diminishment of thereoccurrence of disease, and remission (whether partial or total),whether detectable or undetectable. “Treating” and “treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. “Treating” and “treatment” as used herein alsoinclude prophylactic treatment. For example, a subject with a skindisease, disorder or condition can be treated to prevent progression.Treatment methods comprise administering to a subject a therapeuticallyeffective amount of one or more of the compounds of the disclosure andoptionally consist of a single administration, or alternatively comprisea series of administrations.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” means an amount effective, at dosages and for periodsof time necessary to achieve a desired result. The terms “to treat”,“treating” and “treatment” as used herein and as is well understood inthe art, means an approach for obtaining beneficial or desired results,including clinical results. “To treat”, “treating” and “treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. “To treat”, “treating” and “treatment” as usedherein also include prophylactic treatment.

Where features or aspects of the disclosure are described in terms ofMarkush groups, those skilled in the art will recognize that thedisclosure is also thereby described in terms of any individual memberor subgroup of members of the Markush group.

Further, the definitions and embodiments described in particularsections are intended to be applicable to other embodiments hereindescribed for which they are suitable as would be understood by a personskilled in the art. For example, in the following passages, differentaspects are defined in more detail. Each aspect so defined may becombined with any other aspect or aspects unless clearly indicated tothe contrary. For example, any combination of members of any group canbe combined and optionally combined with any other subgroup of members.In particular, any feature indicated as being preferred or advantageousmay be combined with any other feature or features indicated as beingpreferred or advantageous.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges disclosed herein also encompass any and all possible subrangesand combinations of subranges thereof. Any listed range can be easilyrecognized as sufficiently describing and enabling the same range beingbroken down into at least equal halves, thirds, quarters, fifths,tenths, etc. As a non-limiting example, each range discussed herein canbe readily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art all languagesuch as “up to,” “at least,” “greater than,” “less than,” and the like,include the number recited and refer to ranges which can be subsequentlybroken down into subranges as discussed above. Finally, as will beunderstood by one skilled in the art, a range includes each individualmember.

II. Compositions of the Disclosure

The Applicant has shown that biphasic phospholipid vesicles havingphospholipid bilayers that sequester a stabilized oil-in-water emulsionand a compound which include one or more penetration enhancing agentsadded to the phospholipid bilayers or the stabilized oil-in-wateremulsion or both parts of the delivery system (e.g. compositions and/orother products comprising the biphasic vesicles described herein)provide enhanced skin penetration of the compound.

The Applicant has shown that certain penetration enhancing agents andcombinations of penetration enhancing agents and compounds, relative toother combinations, can be used to more effectively deliver a higherquantity of the compound (e.g. in milligrams) into a quantity of skin(e.g. in grams).

The penetration enhancing agents compounds can be chosen from a widevariety of compounds generally known as penetration enhancers bythemselves. In an embodiment, the Applicant has shown that penetrationenhancing agents such as non-ionic surfactants having ahydrophilic-lipophilic balance (“HLB”) of 10 or less or alone orcombination of with one or more penetration enhancing agents such asterpenes, alkaloids, salicylate derivatives, polycationic (e.g.dicationic, tricationic etc) surfactants such as gemini cationicsurfactants or polycationic amino acids, or combinations thereof provideenhanced skin penetration of the compound compared to an otherwise sameor similar composition except in the absence of the one or morepenetration enhancing agents.

In another embodiment, the Applicant has shown polycationic surfactantssuch as such as gemini dicationic surfactants or polycationic aminoacids enhance skin penetration of the compound relative to otherwisesame or similar composition except with a monocationic surfactant inplace of the polycationic surfactant.

Accordingly, the present application includes a biphasic lipid vesiclecomposition comprising:

-   -   a) lipid vesicles each comprising a lipid bilayer comprising        vesicle forming lipids,    -   b) an oil-in-water emulsion entrapped in the biphasic lipid        vesicles, and stabilized by one or more surfactants;    -   c) one or more compounds entrapped in the lipid bilayer or the        oil-in-water emulsion of the biphasic vesicles); and    -   d) one or more penetration enhancing agents entrapped in the        lipid bilayer or the oil-in-water emulsion of the biphasic        vesicles,

wherein the one or more penetration enhancing agents are one or morenon-ionic surfactants having a hydrophilic-lipophilic balance (HLB) ofabout 10 or less.

In an embodiment, the biphasic lipid vesicle composition is a cosmeticcomposition. In an embodiment, the biphasic lipid vesicle composition isa pharmaceutical composition.

In an embodiment, a pharmaceutical composition (described herein as alipid vesicle composition) is provided for the topical administration ofa therapeutic compound to achieve topical delivery, the compositioncomprising: a lipid vesicle; an oil-in-water emulsion; the therapeuticcompound; and one or more penetration enhancing agents; wherein thelipid vesicle comprises an exterior lipid bilayer; the oil-in-wateremulsion is coated by the exterior lipid bilayer; the therapeuticcompound is for example, a small molecule peptide or protein; and theone or more penetration enhancing agents increases a quantity of thetherapeutic compound that absorbs into a quantity of skin relative tothe composition in the absence of the one or more penetration enhancingagents.

The Applicant has shown that the lipid vesicles can be formulated tohave the compound, and/or the penetration enhancing agents, selectivelyincorporated into the lipid bilayers and/or the oil-in-water emulsion atdifferent stages of production of the biphasic lipid vesicles. Thecompound, for example, can be added only to the oil-in-water emulsion,only to the components of the lipid bilayers, or to both theoil-in-water emulsion and the lipid bilayers during production of thebiphasic lipid vesicles. Similarly, the one or more penetrationenhancing agents, can be added to only to the oil-in-water emulsion,only to the lipid bilayers, or to both the oil-in-water emulsion and thelipid bilayers during production of the biphasic lipid vesicles.

In an embodiment, the biphasic lipid vesicle composition is for thetopical delivery of the one or more compounds. In an embodiment, thetopical delivery is for intradermal, transdermal, mucosal ortransmucosal delivery.

In an embodiment, the biphasic lipid vesicle composition comprises asuspension of the biphasic lipid vesicles.

In an embodiment, the one or more penetration enhancing agents areentrapped in the oil-in-water emulsion of the biphasic lipid vesicle. Inan embodiment, the oil-in-water emulsion of the biphasic lipid vesiclescomprises about 0.01 wt % to about 20 wt % of one or more penetrationenhancing agents. In an embodiment, the oil-in-water emulsion of thebiphasic lipid vesicle comprises about 0.1 wt % to about 10 wt % of oneor more penetration enhancing agents. In an embodiment, the oil-in-wateremulsion of the biphasic lipid vesicle comprises about 0.5 wt % to about9 wt %, about 0.5 wt % to about 8 wt %, about 0.5 wt % to about 7 wt %,about 1 wt % to about 6 wt %, about 1 wt % to about 5 wt %, about 1 wt %to about 4 wt %, about 1 wt % to about 3 wt %, or about 1 wt % to about2 wt %, of one or more penetration enhancing agents.

In an embodiment, the one or more penetration enhancing agents areentrapped in the lipid bilayer of the lipid vesicle. In an embodiment,the lipid bilayer of the lipid vesicle composition comprises 0.1 wt % to20 wt % of the one or more penetration enhancing agents. In anembodiment, the lipid bilayer comprises 0.1 wt % to 10 wt % of the oneor more skin penetration enhancing agents. In an embodiment, the lipidbilayer of the biphasic lipid vesicle comprises about 7 wt % of one ormore skin penetration enhancing agents. In an embodiment, the lipidbilayer of the lipid vesicle comprises about 10 wt %, about 9 wt %,about 8 wt %, about 7 wt %, about 6 wt %, about 5 wt %, about 4 wt %,about 3 wt %, about 2 wt % , about 1 wt %, about 0.5 wt % or about 0.1wt % of one or more skin penetration enhancing agents.

In an embodiment, the one or more penetration enhancing agents areentrapped in both the lipid bilayer and the oil-in-water emulsion of thebiphasic lipid vesicle.

In an embodiment, the penetration enhancing agents are one or morenon-ionic surfactants having a hydrophilic-lipophilic balance (HLB) ofabout 10 or less selected from one or more of polyethylene glycol ethersof fatty alcohols, sorbitan esters, polysorbates, sorbitan esters andpolyethylene glycol fatty acid esters and combinations thereof.

In an embodiment, the polyethylene glycol ethers of fatty alcohols areselected from Ceteth-2®, Steareth-2®, Oleth 2®, Oleth-3®, and Oleth-5®and combinations thereof. In an embodiment, the polyethylene glycolethers of fatty alcohols are selected from Oleth 2®, Oleth-3®, andOleth-5®. In an embodiment, the polyethylene glycol ethers of fattyalcohols is Oleth 2®.

In an embodiment, the sorbitan esters are selected from sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trioleate, sorbitan sesquioleate, and sorbitanIsostearate, and combinations thereof. In an embodiment, the sorbitanesters are selected from sorbitan monolaurate sorbitan monopalmitate,and sorbitan monostearate, and combinations thereof. In an embodiment,the sorbitan esters is sorbitan monopalmitate.

In an embodiment, the polyethylene glycol fatty acid esters are selectedfrom one or more PEG-8 dilaurate, PEG-4 dilaurate, PEG-4 laurate, PEG-8dioleate, PEG-8 distearate, PEG-8 distearate, PEG-7 glyceryl cocoate,and PEG-20 almond glycerides and combinations thereof. In an embodiment,the polyethylene glycol fatty acid esters are selected from PEG-4dilaurate, and PEG-4 laurate and combinations thereof. In an embodiment,the polyethylene glycol fatty acid esters is PEG-4 dilaurate.

In an embodiment, the one or more non-ionic surfactants having ahydrophilic-lipophilic balance (HLB) of about 10 or less are furtherselected from propylene glycol isostearate, glycol stearate, glycerylstearate, glyceryl stearate SE, glyceryl laurate, glyceryl caprylate,PEG-30 dipolyhydroxy-stearate, glycol distearate and combinationsthereof.

In an embodiment, the one or more non-ionic surfactants having ahydrophilic-lipophilic balance (HLB) of about 10 or less are selectedfrom the surfactants in in Table 1:

TABLE 1 Category INCI/Chemical name Properties Ceteth-2 ®(Diethyleneglycol hexadecyl ether) HLB = 5.3 Steareth-2 ®(2-(2-octadecoxyethoxy)ethanol) HLB = 4.9 Oleth-2 ® (Polyoxyethylene (2)Oleyl HLB = 4.9 Ether/Diethylene glycol monooleyl ether)Oleth-3 ®(Polyoxyethylene (3) Oleyl Ether) HLB = 6.6Oleth-5 ®(Polyoxyethylene (5) Oleyl Ether) HLB = 9 Polysorbate 61 ® HLB= 9.6 Sorbitan monolaurate HLB = 8.6 Sorbitan monopalmitate HLB = 6.7Sorbitan monostearate HLB = 4.7 Sorbitan monooleate HLB = 4.3 Sorbitantrioleate HLB = 1.8 Sorbitan sesquioleate HLB = 3.7 Sorbitan IsostearateHLB = 4.7 PEG-8 dilaurate HLB = 10 PEG-4 dilaurate HLB = 6(Polyoxyethylene (8) dilaurate) PEG-4 laurate HLB = 9 ((Polyoxyethylene(4) dilaurate) PEG-8 dioleate HLB = 7.2 PEG-8 distearate HLB = 8 PEG-7glyceryl cocoate HLB = 10 PEG-20 almond glycerides HLB = 10 Propyleneglycol isostearate HLB = 2.5 Glycol stearate HLB = 2.9 Glyceryl stearateHLB = 3.8 Glyceryl stearate SE HLB = 5.8 Glyceryl laurate HLB = 5.2Glyceryl caprylate HLB = 5-6 PEG-30 dipolyhydroxy-stearate HLB = 5.5Glycol distearate HLB = 1, and Phospholipid/lecithin HLB = 4-10and combinations thereof.

In an embodiment, the one or more penetration enhancing agents are oneor more non-ionic surfactants having a HLB of about 10 or less incombination with one or more penetration enhancing agents selected fromone or more terpenes, alkaloids, salicylate derivatives, and di- orpolycationic surfactants and combinations thereof.

In an embodiment, the one or more non-ionic surfactants having a HLB ofabout 10 or less are as described above.

In an embodiment, the one or more terpenes are selected from one or moreeugenol, d-limonene, menthol, menthone, farnesol, neridol, camphor,nerol and thymol, and combinations thereof. In an embodiment, the one ormore terpenes are selected from one or more of menthol, camphor, neroland thymol, and combinations thereof.

In an embodiment, the one or more salicylate derivatives is selectedfrom ethyl salicylate, salicylic acid, acetylsalicylic acid andtrolamine salicylate. In an embodiment, the salicylate derivative ismethyl salicylate.

In an embodiment, the one or more alkaloids are selected from piperidinederivatives (e.g., piperine and lobeline), purine derivative (e.g.,caffeine, theobromine and theophylline), pyridine derivative (e.g.,nicotine), colchicine, pyrrolidine derivative (e.g., N-methylpyrrolidone and hygrine), benzylamine (e.g., capsaicin), isoquinolinederivative (e.g., berberine and sanguinarine) or an imidazole derivative(e.g., histamine and pilocarpine). In an embodiment, the one or morealkaloids are piperidine derivatives. In an embodiment, the one or morealkaloids are piperine or lobeline, or combinations thereof. In anembodiment, the one or more alkaloids is piperine.

In an embodiment, the polycationic surfactants are one or more geminisurfactants.

A gemini surfactant is a surfactant molecule which contains more thanone hydrophobic tail. Each hydrophobic tail has a hydrophilic head(Menger and Keiper, 2000; Kirby et al., 2003). The hydrophobic tails orhydrophilic heads are linked together by a spacer. The hydrophobic tailscan be identical or differ. Likewise, the hydrophilic heads can beidentical or differ. Further, the hydrophilic heads may be anionic (e.g.of a phosphate, sulphate or carboxylate type), cationic (e.g. of aquaternary ammonium type), or neutral (e.g. of a polyether, peptide orsugar type) (Menger and Keiper, 2000). In aqueous solutions, geminisurfactants spontaneously aggregate into micelles whose shape and sizeare particularly sensitive to the length and hydrophobic or hydrophilicnature of the spacer. The spacer can be variable, namely short (e.g., 2methylene groups) or long (e.g., more than 12 methylene groups); rigid(e.g., stilbene) or flexible (e.g., methylene chain); and polar (e.g.,polyether, ethoxyl or polyethoxyl) or nonpolar (e.g., aliphatic,aromatic) (Menger and Keiper, 2000). As the hydrophobic tails,hydrophilic heads and spacer can vary with regard to the above aspects,innumerable different molecules can be designed.

In an embodiment, the type of hydrophobic tail is a 03-030 alkyl group,linear or branched, saturated or unsaturated. In an embodiment, thehydrophilic heads may be anionic, cationic or neutral. In an embodiment,the hydrophilic heads are cationic.

In an embodiment, the gemini surfactants anionic, cationic or neutral.In an embodiment, the polycationic surfactants are one or more geminidicationic surfactants.

In an embodiment, the gemini surfactants comprise a linear hydrocarbontailgroups and quaternary ammonium headgroups. The general structure ofone type of gemini cationic surfactant includes a head group composed oftwo positively charged nitrogen atoms, separated by a spacer (n) of 3,4, 6, 8, 10, 12, or 16 carbon atoms and each containing two methylgroups, and the tails consist of two saturated 12 or 16 carbon atomchains (m=10 or14), respectively.

In an embodiment, the one or more gemini dicationic surfactants are of aquaternary ammonium type. In an embodiment, the one or more geminidicationic surfactants are selected from the group consisting of12-7NH-12, 12-7NCH₃-12, 16-3-16, 12-4(OH)₂-12, and 12-EO1-12. In anembodiment, the one or more gemini cationic surfactants are selectedfrom the group consisting of 12-7NH-12, 12-7NCH3-12, and 16-3-16.

In an embodiment, the one or more polycationic surfactants arepolycationic amino acids. In an embodiment, the polycationic amino acidsare selected from polylysine, polyarginine and combinations thereof.

In an embodiment, the one or more penetration enhancing agents are oneor more non-ionic surfactants having a HLB of about 10 or less incombination with one or more penetration enhancing agents selected fromone or more terpenes, alkaloids, and salicylate derivatives.

In an embodiment, the biphasic lipid vesicle composition comprises oneto six penetration enhancing agents. In an embodiment, the biphasiclipid vesicle composition comprises one to four penetration enhancingagents. In an embodiment, the biphasic lipid vesicle compositioncomprises one to three penetration enhancing agents.

In an embodiment, the penetration enhancing agents are one or morenon-ionic surfactants having a HLB of about 9 or less, about 8 or less,about 7 or less, or about 6 or less and optionally having a HLB of 1 ormore, 2 or more, 3 or more or 4 or more or any combination thereof e.g.about 7 or less and about 3 or more. In an embodiment, the penetrationenhancing agents are one or more non-ionic surfactants having a HLB ofabout 1 to about 10, about 1 to about 9, about 2 to about 8, about 3 toabout 7, or about 4 to about 7. In an embodiment, the penetrationenhancing agents are one or more non-ionic surfactants having ahydrophilic-lipophilic balance (HLB) of, about 3 to about 7, or about 4to about 7. In an embodiment, the penetration enhancing agents are oneor more non-ionic surfactants having a HLB of about 4 to about 7.

In an embodiment, the penetration enhancing agent is Oleth-2®(diethylene glycol monooleyl ether). In an embodiment, the penetrationenhancing agents are Oleth-2® in combination with one or more terpenes.In an embodiment, the penetration enhancing agents are Oleth-2® incombination with one or more of menthol, camphor, nerol or thymol, orcombinations thereof. In an embodiment, the penetration enhancing agentsare Oleth-2® in combination with menthol, or camphor or combinationsthereof. In an embodiment, the penetration enhancing agents are Oleth-2®in combination with menthol and camphor. In an embodiment, thepenetration enhancing agents are Oleth-2® in combination with nerol. Inan embodiment, the penetration enhancing agents are Oleth-2® incombination with thymol. In an embodiment, the penetration enhancingagents are Oleth-2® in combination with nerol. In an embodiment, thepenetration enhancing agents are Oleth-2® in combination with methylsalicylate. In an embodiment, the penetration enhancing agents areOleth-2® in combination with one or more alkaloids. In an embodiment,the penetration enhancing agents are Oleth-2® in combination withpiperidine.

In an embodiment, the one or more non-ionic surfactants having a HLB ofabout 10 or less is entrapped in the lipid bilayer, and the one or moreterpenes or the one or more alkaloids are entrapped in the lipidbilayer, the oil-in-water emulsion or both.

In an embodiment, the one or more penetration enhancing agent is PEG-4dilaurate. In an embodiment, the one or more penetration enhancingagents are PEG-4 dilaurate in combination one or more alkaloids. In anembodiment, the one or more penetration enhancing agents are PEG-4dilaurate in combination with piperidine. In an embodiment, the one ormore penetration enhancing agents are PEG-4 dilaurate in combinationwith methyl salicylate.

In an embodiment, the PEG-4 dilaurate is entrapped in the lipid bilayer,and the one or more alkaloids or the methyl salicylate are entrapped inthe lipid bilayer, the oil-in-water emulsion or both.

In an embodiment, the one or more penetration enhancing agents areOleth-2, PEG-4 dilaurate or sorbitan monopalmitate, or combinationsthereof. In an embodiment, the one or more penetration enhancing agentsare Oleth-2 and sorbitan monopalmitate in combination. In an embodiment,the one or more penetration enhancing agents are PEG-4 dilaurate andsorbitan monopalmitate in combination.

In an embodiment, the Oleth-2®, PEG-4 dilaurate or sorbitanmonopalmitate, or combinations thereof are entrapped in the lipidbilayer, the oil-in-water emulsion or both.

In an embodiment, the one or more penetration enhancing agents increasesa quantity of a compound that absorbs into a quantity of skin by atleast 10%, 20%, 30%, 40%, or 50% relative to an otherwise same orsimilar composition except in the absence of the one or more penetrationenhancing agents. In an embodiment, the one or more penetrationenhancing agents increases a quantity of a compound that absorbs into aquantity of skin by at least about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45% or about 50% relative to anotherwise same or similar composition except in the absence of the oneor more penetration enhancing agents.

In an embodiment, biphasic lipid vesicle comprises from about 0.1 wt %to about 5 wt % of the alkaloid. In an embodiment, biphasic lipidvesicle comprises from about 0.1 wt % to about 4 wt % of the alkaloid.In an embodiment, the biphasic lipid vesicle comprises from about 0.1 wt% to about 3 wt % of the alkaloid. In an embodiment, the biphasic lipidvesicle comprises from about 1 wt % to about 3 wt % of the alkaloid. Inan embodiment, the lipid bilayer of the lipid vesicle comprises from 1wt % to 5 wt % of the alkaloid. In some embodiments, the alkaloid isentrapped in the lipid bilayer of the biphasic lipid vesicle.

Generally, the biphasic lipid vesicle is a multilamellar lipid vesicle,further comprising one or more interior lipid bilayers. Themultilamellar biphasic lipid vesicles that have multiple concentriclipid bilayer shells that encapsulate an oil-in-water emulsion.

In an embodiment, the oil-in-water emulsion includes droplets having anaverage diameter of less than 1 μm. In an embodiment, the averagediameter of the oil-in-water emulsion droplets may be less than 0.5 μm,0.25 μm, 0.1 μm or 0.01 μm. In an embodiment, the average diameter ofthe oil-in-water emulsion droplets may be less than about 0.5 μm, lessthan about 0.25 μm, less than about 0.1 μm or less than about 0.01 μm.Because the oil-in-water emulsion includes aqueous and non-aqueousregions these submicron oil-in-water emulsion droplets can be tuned toincorporate hydrophilic and hydrophobic compounds and excipients.

In an embodiment, the oil-in-water emulsion comprises from 40wt % to99.9 wt % water. In an embodiment, the oil-in-water emulsion includes 10wt % to 95 wt % water, such as 10 wt % to 25 wt %, 25 wt % to 50 wt %,50 wt % to 75 wt %, 75 wt % to 95 wt % water. In an embodiment, theoil-in-water emulsion comprises from about 10wt % to about 99.9 wt %water, from about 15wt % to about 99.9 wt % water, from about 25wt % toabout 99.9 wt % water, from about 25 wt % to about 50 wt % water, fromabout 40 wt % to about 99 wt % water, from about 50wt % to about 95wt %water, from about 50wt % to about 75 wt % water, from about 75wt % toabout 95 wt % water.

In an embodiment, the oil-in-water emulsion comprises from 0.1 wt % to60 wt % of an oil. In an embodiment, the oil-in-water emulsion comprisesfrom about 0.1 wt % to about 60 wt % of an oil, from about 0.5 wt % toabout 50 wt % of an oil, from about 1 wt % to about 40 wt % of an oil orfrom about 1 wt % to about 20 wt % of an oil.

In an embodiment, the oil-in-water emulsion may account for up to about95 wt % of the biphasic lipid vesicle. In other words, in an embodiment,the biphasic lipid vesicle comprises from about 1 wt % to about 95 wt %of the oil-in-water emulsion. In an embodiment, the lipid vesiclecomposition may include 1 wt % to 10 wt %, 20 wt % to 30 wt %, 30 wt %to 40 wt %, 40 wt % to 95wt % of the oil-in-water emulsion. In anembodiment, the lipid vesicle comprises from about 1 wt % to about 10 wt%, from about 20 wt % to about 30 wt %, from about 30 wt % to about 40wt %, from about 40 wt % to about 95wt %, from about 50wt % to about95wt %, from about 60 wt % to about 95wt % or from about 70 wt % toabout 95wt % of the oil-in-water emulsion.

In an embodiment, the oil in the oil-in-water emulsion is selected fromthe group consisting of vegetable oils, mono-, di- and triglycerides,silicone fluids and mineral oils, and combinations thereof. It would beappreciated that the oil-in-water emulsion can be adjusted to havevarious quantities of water and oil to optimize the solubility of anygiven compound, compound, penetration enhancer compounds, surfactantsand/or emulsifiers, etc.

The oil-in-water emulsion of the biphasic lipid vesicles is stabilizedby one or more surfactants. In an embodiment, the oil-in-water emulsionof the biphasic lipid vesicles comprises from 0.01 wt % to 40 wt % ofthe one or more surfactants. Without being bound by theory, it iscontemplated that the surfactants can be added to the oil-in-wateremulsion to modify the stability of the oil-in-water emulsion. In anembodiment, the water-in-oil emulsion comprises 0.01 wt % to 10 wt %, 10wt % to 20 wt % or 20 wt % to 40 wt % of the one or more surfactants. Inan embodiment, the water-in-oil emulsion comprises about 0.01 wt % toabout 40 wt %, about 0.01 wt % to about 10 wt %, about 10 wt % to about20 wt %, about 20 wt % to about 30 wt % about 20 wt % to about 40 wt %,or about 30 wt % to about 40 wt % of the one or more surfactants.

In an embodiment, the oil-in-water emulsion of the biphasic lipidvesicles is stabilized by one or more surfactants selected from thegroup consisting of a polyethylene glycol ether of a fatty alcohol,polyethylene glycol fatty acid ester, polysorbate and a sorbitan ester.In an embodiment, the one or more surfactants have an averagehydrophilic-lipophilic balance (HLB) number greater than 10 or more. Inan embodiment, the one or more surfactants in the oil-in-water emulsionhave a HLB of greater than 10 or more, about 11 or more, about 12 ormore, about 13 or more, about 14 or more, about 15 or more, about 16 ormore, about 17 or more, about 18 or more, about 19 or more or about 20or more or combinations thereof. In an embodiment, the one or moresurfactants in the oil-in-water emulsion have a HLB of greater than 10to about 20, about 10 to about 18, about 10 to about 16, or about 10 toabout 15. In an embodiment, the one or more surfactants in theoil-in-water emulsion have a HLB of about 10 to about 16.In anembodiment, the one or more surfactants in the oil-in-water emulsionhave a HLB of , 10-20 or 10-16.

In an embodiment, the one or more non-ionic surfactants having ahydrophilic-lipophilic balance (HLB) greater than 10 or more areselected from the surfactants in in Table 2:

TABLE 2 Trade name INCI/Chemical name Properties Polyethylene glycolethers of fatty alcohols BRIJ ™ 35Brij ™ L23 Laureth-23 (Polyoxyethylene(23) lauryl ether) HLB = 17.0 Brij 56/Brij ™ C10 Ceteth-10(polyoxyethylene (10) cetyl ether) HLB 12.9 BRIJ ™ 58/Brij ™ C20Ceteth-20 (polyoxyethylene (20) cetyl ether) HLB 15.7 BRIJ ™ 700Steareth-100 (polyoxyethylene (100) stearyl ether) HLB = 18.8 BRIJ ™ 721Steareth-21 (polyoxyethylene (21) stearyl ether) HLB = 15.5 BRIJ ™ 76Steareth-10 (polyoxyethylene (10) stearyl ether) HLB = 12.4 BRIJ ™ 78Steareth-20 (polyoxyethylene (20) stearyl ether) HLB = 15.3 Brij ™ CS20Ceteareth-20 HLB = 15.2 Brij ™ IC20 Isoceteth-20 HLB = 15.7 Brij97Brij ™ O10 Oleth-10 HLB = 12.4 Brij 98Brij ™ O20 Oleth-20 HLB = 15.3Polysorbates Tween20 Polysorbate 20 HLB = 16.7 Tween 21 Polysorbate 21HLB = 13.3 Tween 40 Polysorbate 40 HLB = 15.6 Tween 60 Polysorbate 60 NFHLB = 14.9 Tween 80 Polysorbate 80/ HLB = 15 polyoxyethylene 20 sorbitanmonooleate Tween 85 Polysorbate 85 HLB = 11 Polyethylene glycol fattyacid esters Lipopeg 4-L PEG-8 laurate HLB = 13 Lipopeg 4-S/Myrj 45 PEG-8stearate HLB = 11.2 Lipopeg 10-S/Myrj 49 PEG-20 stearate HLB = 15.2Lipopeg 39-S/Myrj 52 PEG-40 stearate HLB = 16.9 Lipopeg 100-S/Myrj 59PEG-100 stearate HLB = 18.8 Lipopeg 6000-DS PEG-150 distearate HLB =18.4 PEG-25 Hydrogenated HLB = 10.8 Castor Oil PEG-7 Olivate HLB = 11PEG-8 Oleate HLB = 11.6 Stearamide MEA HLB = 11 Cetearyl Glucoside HLB =11 Polyglyceryl-3 HLB = 12 Methyglucose Distearate Cocamide MEA HLB =13.5 Isosteareth-20 HLB = 15 PEG-60 Almond Glycerides HLB = 15Laureth-23 HLB = 16.9 PEG-100 Stearate HLB = 18.8 Steareth-100 HLB =18.8 PEG-80 Sorbitan Laurate HLB = 19.1

In an embodiment, oil-in-water emulsion of the biphasic lipid vesiclesis stabilized by one or more surfactants selected from Ceteth-10® andTween 80® (polysorbate 80 (glycol)/polyoxyethylene 20 sorbitanmonooleate).

The one or more non-ionic surfactants having a hydrophilic-lipophilicbalance (HLB) of about 10 or less of the penetration enhancing agents isnot employed for the stabilization and emulsification of theoil-in-water emulsion, but rather the one or more non-ionic surfactantshaving a hydrophilic-lipophilic balance (HLB) of about 10 or less of thepenetration enhancing agents is used as an additional surfactant to thestabilizing surfactant to provide the permeation enhancing effect.

It would also be appreciated that relative to known biphasic vesiclecompositions where the lipid vesicles contained a surfactant as astabilizing structural ingredient for the creation of oil-in-wateremulsion, the present disclosure uses one or more penetration enhancingagents that, when incorporated into the vesicle structure (either lipidbilayer or oil-in-water emulsion) provide enhanced delivery capabilitiesfor a range of compounds.

In an embodiment, the oil-in-water emulsion comprises from 10 wt % to 99wt % water, from 0.5 wt % to 60 wt % oil and further comprise from 0.01wt % to 20 wt % of one or more surfactants for stabilizing theoil-in-water emulsion .

In an embodiment, the vesicle forming lipids are amphipathic lipidshaving a hydrophobic tail and a head group which can form spontaneouslyinto bilayer vesicles in water. In an embodiment, the vesicle-forminglipids comprise two hydrocarbon chains, such as acyl chains, where thehead group is either polar or nonpolar. In an embodiment, the vesicleforming lipids are selected from one or more of phospholipids,glycolipids, lecithins, and ceramides such as phosphatidylethanolamine,lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid, andcerebroside. These lipids can be obtained commercially or preparedaccording to published methods.

In an embodiment, the vesicle forming lipids are phospholipids. In anembodiment, the phospholipids are one or more esters of glycerol withone or two (equal or different) residues of fatty adds and withphosphoric acid, wherein the phosphoric acid residue is in turn bound toa hydrophilic group, such as, for instance, choline(phosphatidylcholines—PC), serine (phosphatidylserines—PS), glycerol(phosphatidylglycerols--PG), ethanolamine(phosphatidylethanolamines—PE), or inositol (phosphatidylinositol).Esters of phospholipids with only one residue of fatty acid aregenerally referred to in the art as the “lyso” forms of the phospholipidor “lysophospholipids”. Fatty acids residues present in thephospholipids are in general long chain aliphatic acids, typicallycontaining 12 to 24 carbon atoms, or 14 to 22 carbon atoms; thealiphatic chain may contain one or more unsaturations or is completelysaturated. Examples of suitable fatty acids included in thephospholipids are, for instance, lauric acid, myristic acid, palmiticacid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleicacid, and linolenic acid. Saturated fatty acids such as myristic acid,palmitic acid, stearic acid and arachidic acid may be employed.

In an embodiment, the phospholipids are phosphatidic acids, i.e., thediesters of glycerol-phosphoric acid with fatty acids; sphingolipidssuch as sphingomyelins, i.e., those phosphatidylcholine analogs wherethe residue of glycerol diester with fatty acids is replaced by aceramide chain; cardiolipins, i.e., the esters of1,3-diphosphatidylglycerol with a fatty acid; glycolipids such asgangliosides GM1 (or GM2) or cerebrosides; glucolipids; sulfatides andglycosphingolipids.

In an embodiment the phospholipids are naturally occurring,semisynthetic or synthetically prepared products that can be employedeither singularly or as mixtures. In an embodiment, the naturallyoccurring phospholipids are natural lecithins (phosphatidylcholine (PC)derivatives) such as, typically, soya bean or egg yolk lecithins.

In an embodiment, the semisynthetic phospholipids are the partially orfully hydrogenated derivatives of the naturally occurring lecithins. Inan embodiment, the phospholipids include fatty acids di-esters ofphosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol,phosphatidic acid, phosphatidylethanolamine, phosphatidylserine or ofsphingomyelin. In an embodiment, the phospholipids are, for instance,dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine(DMPC), dipalmitoyl-phosphatidylcholine (DPPC),diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine(DSPC), dioleoyl-phosphatidylcholine (DOPC),1,2Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC),dipentadecanoyl-phosphatidylcholine (DPDPC),1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),1-palmitoyl-2-oleylphosphatidylcholine (POPC),1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),dilauroylphosphatidylglycerol (DLPG) and its alkali metal salts,diarachidoylphosphatidylglycerol (DAPG) and its alkali metal salts,dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts,dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts,distearoylphosphatidylglycerol (DSPG) and its alkali metal salts,dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts,dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts,dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts,distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid(DAPA) and its alkali metal salts, dimyristoylphosphatidylethanolamine(DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidylethanolamine(DOPE), diarachidoylphosphatidylethanolamine (DAPE),dilinoleylphosphatidylethanolamine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin(DPSP), and distearoylsphingomyelin (DSSP),dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol(DAPI), dimyristoylphosphatidylinositol (DMPI),dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol(DSPI), dioleoyl-phosphatidylinositol (DOPI).

In an embodiment, the, the phospholipid is dioleoylphosphatidylethanolamine (DOPE) phosphatidylethanolamine (cephalin) (PE),phosphatidic acid (PA), phosphatidylcholine (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) orphosphatidylserine (PS).

In an embodiment, the biphasic lipid vesicle of the biphasic lipidvesicle compositions generally comprises 0.1 wt % to 30 wt %phospholipids. In some embodiments, the lipid vesicle comprises 1 wt %to 10 wt %, 10 wt % to 20 wt %, 20 wt % to 30 wt % of the phospholipids.In some embodiments, the biphasic lipid vesicle comprises 9 wt % to 13wt % phospholipids. In some embodiments, the biphasic lipid vesiclecomprises 10 wt % phospholipids. In some embodiments, the biphasic lipidvesicle comprises 12 wt % phospholipids. In some embodiments, thebiphasic lipid vesicle comprises about 1 wt % to about 10 wt %, about 10wt % to about 20 wt %, about 20 wt % to about 30 wt %, about 9 wt % toabout 13 wt % phospholipids, about 13 wt %, about 12 wt %, about 11 wt %%, or about 10 wt % of phospholipids.

In an embodiment, the one or more compounds are entrapped inoil-in-water emulsion of the biphasic lipid vesicle. In an embodiment,the oil-in-water emulsion comprises from 1 ng/g to 1,000 ng/g of thecompound/ oil-in-water emulsion. In an embodiment, the oil-in-wateremulsion comprises from 1 ng/g to 10 ng/g, from 10 ng/g to 100 ng/g orfrom 100 ng/g to 1,000 ng/g of the compound/oil-in-emulsion.

In an embodiment, the oil-in-water emulsion droplets comprise 0.0000001wt % to 0.0001 wt %, 0.0001 wt % to 0. 1 wt %, 0. 1 wt % to 1 wt %, or 1wt % to 10 wt % of the compound. In an embodiment, the oil-in-wateremulsion comprise about 0.0000001 wt % to about 0.0001 wt %, about0.0001 wt % to about 0. 1 wt %, about 0. 1 wt % to about 1 wt %, orabout 1 wt % to about 10 wt % of the compound. In an embodiment, theoil-in-water emulsion comprises from 0.0000001 wt % to 10 wt % of thecompound.

In an embodiment, the one or more compounds are entrapped in the lipidbilayer of the biphasic lipid vesicle. In an embodiment, the lipidbilayers of the lipid vesicle compositions can be formulated to have oneor more compounds. In an embodiment, the lipid bilayer of the lipidvesicle composition comprises 0.0000001 wt % to 10 wt % of the compound.In an embodiment, the lipid bilayer comprises about 0.0000001 wt % toabout 0.0001 wt %, about 0.0001 wt % to about O. 1 wt %, about O. 1 wt %to about 1 wt %, or about 1 wt % to about 10 wt % of the compound. In anembodiment, the lipid bilayer of the lipid vesicle comprises 1 wt % to 3wt % of the compound.

In an embodiment, the one or more compounds are entrapped in both thelipid bilayer and the oil-in-water emulsion of the biphasic lipidvesicle. In an embodiment, the one or more compounds entrapped in thelipid bilayer are the same as the one or more compounds entrapped in theoil-in-water emulsion of the biphasic lipid. In an embodiment, the oneor more compounds entrapped in the lipid bilayer are different from theone or more compounds entrapped in the oil-in-water emulsion of thebiphasic lipid vesicle.

It would be appreciated, for example, that one or more compoundentrapped in the oil-in-water emulsion would have a faster rate ofrelease than the same one or more compounds entrapped in the lipidbilayer.

In an embodiment, the one or more compounds are selected from but notlimited to, small molecules, proteins, peptides, carbohydrates, nucleicacids, vaccine antigens, and/or plant extracts.

In an embodiment, the one or more compound are therapeutic compounds.Therefore, the composition of the disclosure is a pharmaceuticalcomposition.

In an embodiment, the small molecules are prostaglandins, anestheticagents such as ibuprofen and diclofenac, analgesics or sedativesincluding opioids such as, for example, buprenorphine, fentanyl,sufentanil, alfentanil and remifentanil, cardioactive medication,androgenic steroids, estrogens, progestogens, antihistamines antiviralagents, vitamins, anti-inflammatory agents, antifungal agents,corticosteroids, vitamins, anti-infectives, dermatological agents,medication for the treatment of nausea and sickness amino acids, shortpeptides (upto 1000 Da), carbohydrates or natural compounds andcombinations thereof.

In an embodiment, the cardioactive medication is organic nitrates, suchas nitroglycerin, isosorbide dinitrate and/or isosorbide mononitrate,quinidine sulphate, procainamide, thiazides such as bendroflumethiazide,chlorothiazide and/or hydrochlorothiazide, nifedipine, nicardipine,adrenergic blockers such as timolol and/or propranolol, verapamil,diltiazem, captopril, clonidine or prazosine.

In an embodiment, the androgenic steroids are testosterone,methyltestosterone or fluoxymesterone.

In an embodiment, the estrogens are estradiol valerate, equilin,mestranol, estrone, estriol, 17.beta.-ethinylestradiol ordiethylstilbestrol.

In an embodiment, the antihistamines are diphenhydramine,dimenhydrinate, perphenazine, triprolidine, pyrilamine, chlorcyclizine,promethazine, carbinoxamine, tripelennamine, brompheniramine,clorprenaline, terfenadine and/or chlorpheniramine;

In an embodiment, the anti-infectives are antibiotics, includingpenicillin, tetracycline, chloramphenicol, sulfacetamide,sulfamethazine, sulfadiazine, sulfamerazine, sulfamethizole and/orsulfisoxazole; antiviral agents; antibacterial agents such aserythromycin and/or clarithromycin, and/or other anti-infectivesincluding nitrofurazone and the like.

In an embodiment, the dermatological agents are vitamin A and/or vitaminE.

In an embodiment, the medication for the treatment of nausea and/orsickness is chlorpromazine, granisetron, perphenazine, prochlorperazine,promethazine, thiethylperazine, triflupromazine and/or trimeprazine;

In an embodiment, the progestogens are progesterone, 19-norprogesterone,norethindrone, norethindrone acetate, chlormadinone, ethisterone,etonogestrel, medroxyprogesterone acetate, hydroxyprogesterone caproate,norethynodrel, norelgestromin, 17.alpha.-hydroxyprogesterone,dydrogesterone, dimethisterone, ethinylestrenol, norgestrel,demegestone, promegestone and/or megestrolacetate.

in an embodiment, the small molecules are an anti-inflammatory agentselected from the group consisting of: acemetacin, acetamidocaproicacid, bendazac, benoxaprofen, bermoprofen, bucloxic acid, butibufen,cinmetacin, clidanac, clopirac, felbinac, fenbufen, fenclozic acid,fenoprofen, fentiazac, flunoxaprofen, flurbiprofen, ibuprofen,indomethacin, isofezolac, isoxepac, ketoprofen, lonazolac, loxoprofen,metiazinic acid, mofezolac, naproxen, oxaprozin, pirazolac, pirprofen,pranoprofen, protizinic acid, sulindac, suprofen, suxibuzone,tiaprofenic acid, tolmetin, and/or tropesin. bermoprofen, bucloxic acid,isoxepac, ketoprofen, loxoprofen, zaltoprofen, ampiroxicam, bucolome,celecoxib, difenpiramide, mofebutazone, nimesulide, paranyline,parecoxib, parsalmide, piketoprofen, talniflumate, tenidap,terofenamate, valdecoxib, 21-acetoxypregnenolone, alclometasone,betamethasone, alfa-bisabolol, budesonide, clobetasone, cyclosporin,deflazacort, dexamethasone, diflorasone, desonide, desoximetasone,diflorasone, diflucortolone, difluprednate, ditazol, everolimus,fluazacort, fludrocortisone, flumethasone, fluocinolone, fluocinonide,fluocortin butyl, fluocortolone, fluprednidene acetate, glucametacin,halcinonide, halobetasol propionate, halometasone, halopredone acetate,hydrocortisone, ibuproxam, loteprednol etabonate, mazipredone,memetasone, methylprednisolone, mometasone furoate, oxyphenbutazone,perisoxal, pimecrolimus, prednisolone, prednisone, rimexolone,sirolimus, triamcinolone and/or tacrolimus.

In an embodiment, the small molecule is ibuprofen and/or diclofenac.

In an embodiment, the small molecule is a wound healing compound. In anembodiment, the wound healing compound is bosentan. In an embodiment,the small molecule is an antibiotic. In an embodiment, the antibiotic isvancomycin.

In an embodiment, the protein is cytokine or peptide. In an embodiment,the peptide of the pharmaceutical composition has 2-900 amino acids.

In an embodiment, the amino acid, peptide or protein has a molecularweight of 50 Daltons to 300,000 Daltons. In some embodiments, thetherapeutic compound is a carbohydrate or nucleic acid molecule having amolecular weight between 50-5M Daltons.

In an embodiment, the peptides are polypeptides such as insulin,cytokine, vaccine antigen, growth hormone releasing factor, or antibody.In an embodiment, the polypeptide has a molecular weight of 1000 Daltonsto 300,000 Daltons.

As described above, the pharmaceutical compositions described herein, attimes referred to as lipid vesicles or lipid compositions orformulations, can be used to deliver a therapeutic compound, includingbut not limited to small molecules, peptides, proteins,carbohydrates,nucleic acids, vaccine antigens, and/or plant extracts .The lipid vesicle formulations include one or more lipid (e.g.,phospholipid) bilayers that contain an oil-in-water emulsion. Theoil-in-water emulsion includes droplets that are generally less than 1μm within the aqueous interior of the lipid vesicles, which aregenerally multilamellar, having multiple lipid bilayers. The biphasiclipid vesicle formulations may also include one or more other lipidvesicle components including but limited to fatty substances such ascholesterol, penetration enhancers, surfactants, solvents etc. to adaptthe lipid vesicle formulations to suit physicochemical propertiesrelated to the target skin. The therapeutic compound, penetrationenhancers, surfactants and/or other lipid vesicle components can beincorporated into the lipid bilayer and/or within the oil-in-wateremulsion.

In an embodiment, the lipid vesicles can be formulated to havecompounds, penetration enhancing agents, surfactants and/or other lipidvesicle componentselectively incorporated into the lipid bilayers and/orthe oil-in-water emulsion at different stages of production. Thus, asubstantial degree of control can be maintained over the location withinthe lipid vesicles at which the compound, penetration enhancing agents,and/or other lipid vesicle component. are incorporated. The compound,for example, can be added only to the components of the oil-in-wateremulsion, only to the components of the lipid bilayers, or to both theoil-in-water emulsion and the lipid bilayers during production of thelipid vesicles.

The structure and composition of these lipid vesicle formulations can betuned to allow the one or more compound, s to deeply penetrate the skin.The lipid bilayers and oil-in-water emulsion of the lipid vesicleformulations sequester the one or more compounds and otherpharmaceutical excipients to provide enhanced stability and sustainedrelease of the compounds. In an embodiment, the biphasic lipid vesicleformulations optionally further comprises one or more other lipidvesicle components including but limited to fatty substances such ascholesterol, penetration enhancers, surfactants, and solvents, andcombinations thereof.

In an embodiment, the lipid bilayer of the lipid vesicle furthercomprises a fatty substance to, for example, enhance the strength of thelipid bilayer. In an embodiment, the fatty substance is cholesterol,cholesterol derivatives, coprostanol, cholestanol, cholestane, or longchain fatty acids or combinations thereof. In an embodiment, the lipidbilayer of the lipid vesicle composition further comprises 0.1 wt % to10 wt % cholesterol and/or a cholesterol derivative. In someembodiments, the lipid bilayer comprises from 1 wt % to 5 wt %cholesterol and/or a cholesterol derivative.

The lipid bilayer of the lipid vesicle composition may include 0.1 wt %to 5 wt % cholesterol or a derivative thereof. In some embodiments, thelipid bilayer of the lipid vesicle composition comprises 0.1 wt % to 3wt % cholesterol or a derivative thereof. In some embodiments, the lipidbilayer comprises 2 wt % cholesterol or a derivative thereof.

In an embodiment, the lipid bilayer of the lipid vesicle compositionoptionally further comprises one or more penetration enhancers inaddition to the one or more penetration enhancing agents. The skinpenetration enhancers includes any known skin penetration enhancers notincluding the one or more penetration enhancing agents such as thosedescribed by Adrian C. Williams and Brian W. Barry Advanced DrugDelivery Reviews 64 (2012) 128-137; or by Majella E. Lane Int. J. Pharm.447 (2013) 12-21.

It would be appreciated that the one or more additional penetrationenhancers in addition to the penetration enhancing agents describedherein can be added to the formulations.

In some embodiments, the skin penetration enhancer is selected from oneor more of an alcohol such as ethanol or isopropyl alcohol; an amidesuch as azone; an ester such as ethyl acetate, padimate O, ethyl oleate,glyceryl monoleate, glyceryl monocaprate, glyceryl tricaprylate,isopropyl myristate, isopropyl palmitate, propylene glycol monolaurate,or propylene glycol monocaprylate; an ether alcohol such as Transcutol®(e.g., Transcutol P, 2-(2-ethoxyethoxy)ethanol); a fatty acid such aslauric acid, linoleic acid, linolenic acid, myristic acid, oleic acid,palmitic acid, stearic acid, or isostearic acid; a glycol such asdipropylene glycol, propylene glycol, 1,2-butylene glycol, or 1,3-butylene glycol; a pyrrolidone such as N-methyl-2-pyrrolidone or2-pyrrolidone; a sulphoxide such as decylmethyl sulphoxide or dimethylsulphoxide.

In an embodiment, the one or more penetration enhancers are fattyacylated amino acids such as monolauroyllysine and/or dipalmitoyllysine.

In an embodiment, the lipid bilayer optionally further comprises ahydrophilic solvent to, for example, solubilize the vesicle forminglipids. In an embodiment, the hydrophilic solvents include but are notlimited to propylene glycol, glycerol, polyethylene glycol having amolecular weight ranging between 300 and 8000, ethanol, and combinationsthereof.

In an embodiment, the oil-in-water emulsion comprises an aqueous mediumhaving water and, optionally, one or more lipophilic additives, such aspreservatives (parabens, phenoxy ethanols, benzalkonium salts, etc.),antioxidants (ascorbic acid, ascorbyl palmitate, BHA, BHT,a-tocopherol), waxes and viscosity enhancing agents (long chain fattyalcohols and their esters, fatty acids, beeswax, olive oil, glycerylstearate, cetyl alcohol, stearyl alcohol, myristyl myristate, and cetylpalmitate, stearyl heptanoate, and/or stearyl palmitate.

In an embodiment, the oil-in-water emulsion includes 0.1 wt % to 25 wt %of the one or more lipophilic additives.

The Applicant has also shown that penetration enhancing agents such aspolycationic surfactants enhance skin penetration of the compoundrelative to otherwise same or similar composition except with amonocationic surfactant in place of the polycationic surfactants.

Accordingly, the present application further includes a biphasic lipidvesicle composition comprising:

-   -   a) lipid vesicles comprising a lipid bilayer comprising vesicle        forming lipids,    -   b) an oil-in-water emulsion entrapped in the biphasic lipid        vesicles, and comprising one or more polycationic surfactants;        and    -   c) one or more compounds entrapped in the lipid bilayer and/or        the oil-in-water emulsion.

In an embodiment, the biphasic lipid vesicle composition is a cosmeticcomposition. In an embodiment, the biphasic lipid vesicle composition isa pharmaceutical composition.

In an embodiment, the biphasic lipid vesicle composition is for thetopical delivery of the one or more compounds. In an embodiment, thetopical delivery is for intradermal, transdermal and/or transmucosaldelivery.

In an embodiment, the biphasic lipid vesicle composition comprises asuspension of the biphasic lipid vesicles.

In an embodiment, the polycationic surfactants are one or more geminisurfactants.

A gemini surfactant is a surfactant molecule which contains more thanone hydrophobic tail. Each hydrophobic tail has a hydrophilic head(Menger and Keiper, 2000; Kirby et al., 2003). The hydrophobic tails orhydrophilic heads are linked together by a spacer. The hydrophobic tailscan be identical or differ. Likewise, the hydrophilic heads can beidentical or differ. Further, the hydrophilic heads may be anionic (e.g.of a phosphate, sulphate or carboxylate type), cationic (e.g. of aquaternary ammonium type), or neutral (e.g. of a polyether, peptide orsugar type) (Menger and Keiper, 2000). In aqueous solutions, geminisurfactants spontaneously aggregate into micelles whose shape and sizeare particularly sensitive to the length and hydrophobic or hydrophilicnature of the spacer. The spacer can be variable, namely short (e.g., 2methylene groups) or long (e.g., more thanl2 methylene groups); rigid(e.g., stilbene) or flexible (e.g., methylene chain); and polar (e.g.,polyether, ethoxyl or polyethoxyl) or nonpolar (e.g., aliphatic,aromatic) (Menger and Keiper, 2000). As the hydrophobic tails,hydrophilic heads and spacer can vary with regard to the above aspects,innumerable different molecules can be designed.

In an embodiment, the type of hydrophobic tail is a C₃-C₃₀ alkyl group,linear or branched, saturated or unsaturated. In an embodiment, thehydrophilic heads may be anionic, cationic or neutral. In an embodiment,the hydrophilic heads are cationic.

In an embodiment, the polycationic surfactants are one or more geminidicationic surfactants.

In an embodiment, the gemini surfactants comprise a linear hydrocarbontailgroups and quaternary ammonium headgroups. The general structure ofone type of gemini cationic surfactant includes a head group composed oftwo positively charged nitrogen atoms, separated by a spacer (n) of 3,4, 6, 8, 10, 12, or 16 carbon atoms and each containing two methylgroups, and the tails consist of two saturated 12 or 16 carbon atomchains (m=10 or 14), respectively.

In an embodiment, the one or more gemini dicationic surfactants are of aquaternary ammonium type. In an embodiment, the one or more geminidicationic surfactants are selected from the group consisting of12-7NH-12, 12-7NCH₃-12, 16-3-16, 12-4(OH)₂-12, and 12-EO1-12. In anembodiment, the one or more gemini cationic surfactants are selectedfrom the group consisting of 12-7NH-12, 12-7NCH3-12, and 16-3-16.

In an embodiment, the one or more polycationic surfactants arepolycationic amino acids. In an embodiment, the polycationic amino acidsare selected from polylysine, polyarginine and combinations thereof.

In an embodiment, the oil-in-water emulsion of the biphasic lipidvesicles comprises from about 0.01 to about 5%, 0.05 to about 5%, 0.1%to about 5%, about 1% to about 5%, or about 2% to about 5% of the one ormore polycationic surfactants. In an embodiment, the oil-in-wateremulsion of the biphasic lipid vesicles comprises from about 0.01 toabout 5% of the one or more polycationic surfactants.

In an embodiment, the oil-in-water emulsion of the biphasic lipidvesicles optionally comprises one or more additional surfactants (notincluding the polycationic surfactants). In an embodiment, the one ormore additional surfactants are the one or more additional stabilizingsurfactants as described above. In an embodiment, the oil-in-wateremulsion of the biphasic lipid vesicles comprises from 0.1% to about 10%of the one or more surfactants. In an embodiment, the oil-in-wateremulsion of the biphasic lipid vesicles comprises from about 0.01 toabout 10%, 0.05 to about 10%, 0.1% to about 10%, about 1% to about 10%,about 2% to about 10%, 0.01 to about 7%, 0.05 to about 7%, 0.1% to about7%, about 1% to about 7%, about 2% to about 7%, of the one or moresurfactants.

When used with one or more additional surfactants, the oil-in-wateremulsion of the biphasic lipid vesicles comprises from about 0.1% toabout 10% of the one or more polycationic surfactants. In an embodiment,the oil-in-water emulsion of the biphasic lipid vesicles comprises fromabout 0.01 to about 10%, 0.05 to about 10%, 0.1% to about 10%, about 1%to about 10%, about 2% to about 10%, 0.01 to about 7%, 0.05 to about 7%,0.1% to about 7%, about 1% to about 7%, about 2% to about 7%, of the oneor more polycationicsurfactants.

In an embodiment, the biphasic lipid vesicle composition furtherincludes one or more penetration enhancing agents wherein the one ormore penetration enhancing agents are one or more non-ionic surfactantshaving a HLB of about 10 or less alone, or in combination with one ormore penetration enhancing agents selected from one or more terpenes,alkaloids, salicylate derivatives, and polycationic surfactants andcombinations thereof as described above.

In an embodiment, the wt% water and oil in the oil-in-water emulsion isas described above.

In an embodiment, the vesicle forming lipids are as described above.

In an embodiment, the one or more one or more compounds are entrapped inoil-in-water emulsion of the biphasic lipid vesicle, the lipid bilayer.

In an embodiment, the one or more compounds are entrapped in the lipidbilayer, the oil-in-water emulsion of the biphasic lipid vesicle or bothas described above.

In an embodiment, amount of one or more compound in the lipid bilayer,and the oil-in-water emulsion is as described above.

In an embodiment, the one or more compounds are selected from but notlimited to, small molecules including negatively charged smallmolecules, carbohydrates, nucleic acids such as RNA or DNA or hybridsthereof, plasmid DNA, oligonucleotides, including syntheticoligonucleotides, viral DNA, DNA vaccines, and the like, protein,peptides including peptide antigens such as vaccines antigens,immunoglobulins, immunomodulators, hormones, toxins, and/or enzymes, aswell as plant extracts, and/or vitamins.

In an embodiment, the one or more compounds are selected from but notlimited to peptides, carbohydrates, nucleic acids, vaccine antigens,plasmid DNA, DNA vaccines, peptide vaccines, immunoglobulins,immunomodulators, oligonucleotides, hormones, toxins, and enzymes. In anembodiment, the one or more compounds are selected from the nucleicacids, plasmid DNA, DNA vaccines, and/or oligonucleotides. In anembodiment, the one or more compounds are selected from the nucleicacids, plasmid DNA, DNA vaccines, and/or oligonucleotides.

In an embodiment, the biphasic lipid vesicle compositions optionallyfurther comprise one or more other lipid vesicle components includingbut limited to fatty substances such as cholesterol, penetrationenhancers, surfactants, and/or solvents, and combinations thereof asdescribed above.

In an embodiment, the biphasic lipid vesicle compositions of thedisclosures are for the topical delivery of the one or more compounds.In an embodiment, the topical delivery is for intradermal, transdermalor transmucosal delivery.

As noted above, in an embodiment, the biphasic lipid vesiclecompositions of the disclosure described herein can be cosmeticcompositions.

In an embodiment, the biphasic lipid vesicle cosmetic compositions ofthe disclosure suitably optionally comprise components generally used incosmetic products, for example, moisturizers, antioxidants, oilycomponents, UV absorbers, emulsifiers, thickeners, alcohols, powdercomponents, colorants, aqueous components, water, and/or various skinnutrients, etc., as needed, within the range that does not impair theeffect of the present compositions and system. The cosmetic compositionmay contain conventional adjuvants and carriers, such as antioxidants,stabilizers, solubilizers, vitamins, pigments, and/or fragrances.

In an embodiment, the biphasic lipid vesicle compositions of thedisclosure described herein can be formulated as a cream, tonic,ointment, paste, lotion, gel, oil, liquid spray, foundation or powder.

In an embodiment, ointments or creams can be formulated with an aqueousor oily base with the addition of suitable thickening and/or gellingagents. Such bases may include water and/or an oil such as liquidparaffin or a vegetable oil such as peanut oil or castor oil. Anexemplary base is water. Thickening agents which can be used accordingto the nature of the base include aluminum stearate, hydrogenatedlanolin, and the like. Further, lotions can be formulated with anaqueous base and will, in general, include one or more of the following:stabilizing agents, emulsifying agents, dispersing agents, suspendingagents, thickening agents, coloring agents, perfumes, and the like.Ointments and creams can also contain excipients, such as starch,tragacanth, cellulose derivatives, carbopols, polyethylene glycols,silicones, bentonites, Veegum (magnesium aluminium silicate), silicicacid, and talc, or mixtures thereof. Lotions may be formulated with anaqueous or oily base and will, in general, also include one or more ofthe following: stabilizing agents, emulsifying agents, dispersingagents, suspending agents, thickening agents, coloring agents, perfumes,and the like. Foams may be formed with known foaming or surface activeagents.

In an embodiment, the gels may be formed by mixing the delivery system(e.g. the biphasic vesicles described herein) with gelling agents suchas collagen, pectin, gelatin, agarose, chitin, chitosan and alginate.The delivery system may be incorporated into liquids, formulated astopical solutions, aerosols, mists, sprays, drops and instillationsolutions for body cavities. dministration of the delivery system to forexample the mucosal membrane may be performed by aerosol, which can begenerated by a topical aerosol spray pump or actuator, or byinstillation.

Also provided is a container comprising a composition described herein.The container is optionally a spray container optionally an aerosolspray pump container.

In an embodiment, the biphasic lipid vesicle compositions of thedisclosure described herein is comprised in a coated substrate such asdressings, packings, films or meshes which can coated with the biphasiclipid vesicle composition and used directly on the skin or mucosalmembrane.

In an embodiment, the biphasic lipid vesicle compositions of thedisclosure described herein may be comprised in a transdermal deliverysystem taking one of various forms, for example, a patch or a masksheet.

In an embodiment, the transdermal delivery system comprises

a backing layer; and

a matrix layer comprising a biphasic lipid vesicle composition describedherein, disposed on the backing layer,

wherein the matrix layer is configured for contacting skin.

In an embodiment, the backing layer is or comprises a polymer selectedfrom the group consisting of polyesters, such as polyethyleneterephthalates (PET), as well as polycarbonates, polyolefins such as,for example, polyethylenes, polypropylenes or polybutylenes,polyethylene oxides, polyurethanes, polystyrenes, polyamides,polyimides, polyvinyl acetates, polyvinyl chlorides, polyvinylidenechlorides, copolymerisates such as, for example,acrylonitrile-butadiene-styrene terpolymers, or ethylene-vinylacetate-copolymerisates. A preferred material for a backing layer isselected from a polyester, particular preferably from a polyethyleneterephthalate. A backing layer of this type may, for example, beobtained from 3M (USA) under the trade name Scotchpak 1109.

In an embodiment, the backing layer is an occlusive backing layer,

The backing layer can for example be produced from polyesters.

In another embodiment, the backing layer comprises an overtape whichprotrudes laterally beyond the edges of the matrix layer, permittingadhesion or better adhesion of the transdermal delivery system to theskin. The overtape can comprise a layer of adhesive, free from activeingredient and overtape film. The overtape film can be a polymerselected from the group formed by polyolefins, olefin copolymerisates,polyesters, copolyesters, polyamides, copolyamides, polyurethanes andthe like. Examples of suitable materials that may be cited arepolyesters, and of these, polyethylene terephthalates in particular, aswell as polycarbonates, polyolefins such as, for example, polyethylenes,polypropylenes or polybutylenes, polyethylene oxides, polyurethanes,polystyrenes, polyamides, polyimides, polyvinyl acetates, polyvinylchlorides, polyvinylidene chlorides, copolymerisates such as, forexample, acrylonitrile-butadiene-styrene terpolymers, or ethylene-vinylacetate-copolymerisates.

In an embodiment, the adhesive can for example be polyisobutylene (PIB)adhesive.

In an embodiment, the backing layer has a thickness which is at leastabout 5 μm, at least about 10 μm, at least about 15 μm, at least about20 μm, at least about 25 μm, at least about 50 μm, at least about 75 μm,at least about 100 μm, at least about 125 μm, or up to approximately 250μm, up to approximately 200 μm, up to approximately 150 μm, up toapproximately 100 μm or up to 50 μm, or any combination of theforegoing. The backing layer can for example have a thickness includingor between 5 μm and 200 μm or any 0.1 μm increment between 5 μm and200μm.

When the transdermal delivery system is a patch, the backing layerthickness may be at least about 75 μm or at least about 100 μm and lessthan for example 200 μm or less than for example 150 μm

When the transdermal delivery system is a mask, the backing laterthickness may be at least 10 μm or at least 20 μm and less than forexample 100 μm or less than for example 75 pm.

The matrix layer has a surface which is intended to be placed on theskin can be referred to as the application side. The application sidemay be configured so as to comprise a pressure-sensitive adhesive overits entire surface, for example a surface self-adhesive glue or it maybe configured so as to be adhesive over only a portion of its surface.

In an embodiment, the transdermal delivery system further comprises aprotective layer, also known as a release liner, which is applied to thecomposition comprising matrix layer and which is removed prior toapplication of the transdermal delivery system. to facilitate removal ofthe protective layer, in some embodiments, the protective layerprotrudes beyond the edge of the backing layer e.g. the remaining patch.

In an embodiment, the transdermal delivery system is a patch.

In an embodiment, the one or more compounds are therapeutic compounds.Therefore, the biphasic lipid vesicle compositions of the disclosuredescribed herein are pharmaceutical compositions. Accordingly, thebiphasic lipid vesicles of the disclosure are suitably formulated intopharmaceutical compositions for administration to subjects in abiologically compatible form suitable for topical administrationcomprising pharmaceutical acceptable carriers. In an embodiment, the oneor more compounds are therapeutic compounds are selected from the one ormore therapeutic compounds described herein.

III. Methods of Preparing the Compositions of the Disclosure

The compositions of the disclosure as described above are prepared bymixing oil components of the oil-in-water emulsion with aqueouscomponents of the oil-in-water emulsion wherein either the oilcomponents or aqueous components of the oil-in-water emulsion comprisesone or more surfactants for emulsification of the oil component with theaqueous component of the oil-in-water emulsion. In an embodiment, thesurfactant is mixed with the aqueous component and added to the oil forformation of an emulsion. The oil-in-water emulsion is then mixed withthe solubilized vesicle-forming lipid and, if added, other lipidcomponents under mixing conditions effective to form the biphasic lipidvesicles.

The one or more penetration enhancing agents and the one or morecompounds are added to oil component of the oil-in-water emulsion, tothe aqueous component of the oil-in-water emulsion or both.Alternatively, or in addition to, the one or more penetration enhancingagents and the one or more compounds can be added to the lipidcomponent.

Accordingly, the present application includes a method of preparingbiphasic lipid vesicles comprising:

-   -   a) preparing an oil-in-water emulsion comprising one or more        surfactants, by mixing oil components of the oil-in-water        emulsion with aqueous components of the oil-in-water emulsion,        wherein the oil components and/or the aqueous components of the        oil-in-water emulsion comprises the one or more surfactants;    -   b) solubilizing vesicle forming lipids in an acceptable solvent        other than water,    -   c) adding one or more compounds and one or more penetration        enhancing agents to the oil components and/or the aqueous        components of step a), and/or the solubilized vesicle forming        lipids of step b);    -   d) adding the oil-in-water emulsion to the solubilized vesicle        forming lipids; and    -   e) mixing the oil-in-water emulsion and the solubilized vesicle        forming lipids under mixing conditions effective to form the        biphasic lipid vesicles comprising a lipid bilayer comprising        vesicle forming lipids, and an oil-in-water emulsion entrapped        in the biphasic lipid vesicles.

In an embodiment, a pharmaceutical composition, i.e., lipid vesiclecomposition, is provided for the topical administration of a compound,wherein the composition comprises a lipid vesicle comprising an exteriorlipid bilayer, an oil-in-water emulsion and the therapeutic compound,the composition being formed by: (a) mixing oil with water to form theoil-in-water emulsion; (b) mixing the oil-in-water emulsion of (a) withat least one vesicle forming lipid such that the oil-in-water emulsionis coated by the exterior lipid bilayer; and (c) adding the therapeuticcompound and penetration enhancers during (a) and/or (b); wherein thecompound is a molecule having a molecular weight between 50-5M Daltons;and the one or more penetration enhancing agents increases a quantity ofthe compound that absorbs into a quantity of skin relative to the samecomposition in the absence of the one or more penetration enhancingagents.

In an embodiment, the mixing oil components of the oil-in-water emulsionwith aqueous components of the oil-in-water emulsion vesicles of step a)and/or the mixing conditions of step e) comprises using agitation suchas homogenization or emulsification, or micro-emulsion techniques whichdo not involve agitation. In an embodiment, the mixing comprises highpressure homogenizing. The high pressure homogenizing providesrelatively precise control over the composition of the lipid vesicles.High pressure homogenizing is suitable for small molecules and peptidesor proteins that are resistant to shearing. In an embodiment, thecomposition that is formed is any one of the lipid vesicle compositionsdescribed herein.

In an embodiment, other lipid components are added to any one of stepsa) to e).

In an embodiment, the one or more surfactants are selected from one ormore stabilizing surfactants and/or one or more polycationic surfactantsdescribed herein.

In an embodiment, the one or more penetration enhancing agents, the oneor more compounds, the oil-in-water emulsion, the vesicle forming lipid,the acceptable solvent and/or the other lipid components are asdescribed above.

The lipid vesicle compositions of the disclosure can also be prepared bymethods known in the art, for example by the methods disclosed in U.S.Pat. Nos. 5,993,852, 5 ,853,755 and U.S. Pat. No.5,993,851 incorporatedherein by reference.

In an embodiment, the biphasic lipid vesicle compositions of thedisclosure described herein may be comprised in a transdermal deliverysystem taking one of various forms, for example, a patch or a masksheet. In an embodiment, the biphasic lipid vesicle compositions is atransdermal patch.

In an embodiment, a transdermal patch can be prepared using proceduresknown in the transdermal patch art. The process for preparation willgenerally involve formulating the matrix layer comprising the biphasic(i.e., mixing the adhesive and the biphasic lipid vesicles andadditives, if any), casting the matrix layer onto the backing or releaseliner layer, and removing solvent from the matrix

IV. Methods and uses of the Disclosure

The biphasic lipid vesicles are liposomes i.e., microscopic vesiclescomposed of a single phospholipid bilayer or a plurality of concentricphospholipid bilayers which enclose the oil-in-water emulsion. Theselipid vesicles serve as compound carriers for the topical delivery ofcompound that may be hydrophobic or hydrophilic. The lipid vesicles aregenerally biocompatible, biodegradable and non-toxic vehicles for drugdelivery.

The compositions of the disclosures can be used for the topical deliveryof one or more compounds. Accordingly, the present application includesa method of delivering one or more compounds by administering thebiphasic lipid vesicle compositions of the disclosures topically to theskin or mucosal membrane to a subject.

The application also includes a use of the lipid vesicle compositions ofthe disclosures of the disclosure for delivering one or more compoundstopically to the skin or mucosal membrane, as well as a use of the lipidvesicle compositions of the disclosures of the disclosure for thepreparation of a medicament for delivering one or more compoundstopically to the skin or mucosal membrane. The application furtherincludes the lipid vesicle compositions of the disclosures of thedisclosure for delivering one or more compounds topically to the skin ormucosal membrane.

The biphasic lipid vesicle compositions of the disclosure comprising theone or more penetration enhancing agents described herein have beenshown to improve the skin permeation of the one or more compoundsrelative to otherwise same or similar compositions except in the absenceof the one or more penetration enhancing agents. The biphasic lipidvesicle compositions of the disclosure and the biphasic lipid vesiclecosmetic compositions of the disclosure comprising the one or morepolycationic surfactants described herein have been shown to improve theskin permeation of the one or more compounds relative to otherwise sameor similar compositions except with a monocationic surfactant in placeof the di- or polycationic surfactant.

Accordingly, the present application also includes a method of improvingtopical delivery of one or more compounds comprising administering aneffective amount of the biphasic lipid vesicle compositions of thedisclosures of the disclosure to the skin or mucosal membrane of asubject in need thereof.

The application also includes a use of the lipid vesicle compositions ofthe disclosure or the lipid vesicle cosmetic compositions of thedisclosure for improving topical delivery of one or more compounds tothe skin or mucosal membrane, as well as a use of the lipid vesiclecompositions of the disclosure or the lipid vesicle cosmeticcompositions of the disclosure for the preparation of a medicament forimproving topical delivery of one or more compounds to the skin ormucosal membrane. The application further includes the lipid vesiclecompositions of the disclosure or the lipid vesicle cosmeticcompositions of the disclosure for improving topical delivery of one ormore compounds to the skin or mucosal membrane.

In an embodiment, the present application includes a method of treatingor preventing skin conditions related to excessive or defective collagenproduction in a subject comprising administering to the subject in needthereof, an effective amount of the lipid vesicle cosmetic compositionsof the disclosure to a subject in need thereof.

The application also includes a use of the lipid vesicle cosmeticcompositions of the disclosure for treating or preventing s preventingskin conditions related to excessive or defective collagen, as well as ause of the lipid vesicle cosmetic compositions of the disclosure for thepreparation of a medicament for treating or preventing skin conditionsrelated to excessive or defective collagen. The application furtherincludes the lipid vesicle cosmetic compositions of the disclosure fortreating or preventing skin conditions related to excessive or defectivecollagen.

In an embodiment, the skin conditions related to excessive or defectivecollagen is skin aging, skin elasticity, striae, stretchmarks, wrinkles,collagen vascular diseases such as cutaneous scleroderma, morphoea,lupus, rheumatoid arthritis, temporal arteritis, fereditary collagendiseases such as Ehlers-Danlos syndrome, Marfan's syndrome.

In an embodiment, the one or more compound are one or more therapeuticcompounds. Therefore, the biphasic lipid vesicle compositions is abiphasic lipid vesicle pharmaceutical composition.

Accordingly, the present application also includes a method of treatingdisease, disorder or condition treatable by delivering one or moretherapeutic compounds by administering a therapeutically effectiveamount of the biphasic lipid vesicle pharmaceutical compositions of thedisclosure topically to the skin or mucosal membrane to a subject inneed thereof. In an embodiment, the biphasic lipid vesicle compositionsof the disclosure are administered topically to the skin.

The application also includes a use of lipid vesicle compositions of thedisclosure for treating diseases, disorders or conditions treatable bydelivering one or more therapeutic compounds of the disclosure topicallyto the skin or mucosal membrane as well as a use of lipid vesiclecompositions of the disclosure for the preparation of a medicament fortreating diseases, disorders or conditions treatable by delivering oneor more therapeutic compounds topically to the skin or mucosal membraneto a subject in need thereof. The application further includes lipidvesicle compositions the application for treating diseases, disorders orconditions treatable by delivering one or more therapeutic compoundstopically to the skin or mucosal membrane.

In an embodiment, the disease, disorder or condition treatable bydelivering one or more therapeutic compounds by administering atherapeutically effective amount of the biphasic lipid vesiclepharmaceutical compositions of the disclosure topically to the skin ormucosal membrane is skin condition related to excessive or defectivecollagen production, inflammation, pain, a fungal infection, a viralinfection, skin/dermatological conditions, rheumatic conditions, jointconditions, skin aging or cancer. In an embodiment, the disease,disorder or condition is skin aging. . In an embodiment, the disease,disorder or condition is skin condition related to excessive ordefective collagen production.

In an embodiment, the disease, disorder or condition is a skincondition. In an embodiment, the skin condition is scleroderma, atopicdermatitis, psoriasis, conditions characterized by any cytokinedeficiency, conditions characterized by IFNy deficiency, genodermatoses(skin diseases of genetic origin) including epidermal fragilitydisorders, keratinization disorders, hair disorders, pigmentationdisorders, porphyrias, multisystem disorders and cancer disorders. In anembodiment, the disease, disorder or condition is forms of inheritedepidermolysis bullosa (such as junctional EB and dystrophic EB),lamellar ichthyosis and/or X-linked ichthyosis and xerodermapigmentosum.

In an embodiment, the disease, disorder or condition is an infection. Inan embodiment, the infection is a viral infection, a bacterial infectionor fungal infection.

In an embodiment, the disease, disorder or condition is sexualdysfunction. In an embodiment, the sexual dysfunction is erectiledysfunction or impotence.

In an embodiment, the disease, disorder or condition is genetic warts.

In an embodiment, the disease, disorder or condition is pain orinflammation. In an embodiment, the pain is acute pain or chronic pain.

In an embodiment, the subject is a mammal. In an embodiment, the subjectis a human.

The dosage of compositions of the disclosure can vary depending on manyfactors such as the pharmacodynamic properties of the compound, the modeof administration, the age, health and weight of the recipient, thenature and extent of the symptoms, the frequency of the treatment andthe type of concurrent treatment, if any, and the clearance rate of thecompound in the subject to be treated. One of skill in the art candetermine the appropriate dosage based on the above factors.Compositions of the disclosure may be administered initially in asuitable dosage that may be adjusted as required, depending on theclinical response. Dosages will generally be selected to maintain aserum level of compounds of the disclosure from about 0.01 μg/mL toabout 1000 μg/mL, or about 0.1 μg/mL to about 100 μg/mL. Asrepresentative amount is from about 0.001 mg/kg to about 10 mg/kg, about0.1 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 1 mg/kg or about0.1 mg/kg to about 1 mg/kg. Compounds of the disclosure may beadministered in a single daily, weekly or monthly dose or the totaldaily dose may be divided into two, three or four daily doses.

In an embodiment, the compositions of the disclosure are administered atleast once a week. However, in another embodiment, the compounds areadministered to the subject from about one time per two weeks, threeweeks or one month. In another embodiment, the compounds areadministered about one time per week to about once daily. In anotherembodiment, the compounds are administered 2, 3, 4, 5 or 6 times daily.The length of the treatment period depends on a variety of factors, suchas the severity of the disease, disorder or condition, the age of thesubject, the concentration and/or the activity of the compounds of thedisclosure, and/or a combination thereof. It will also be appreciatedthat the effective dosage of the compound used for the treatment mayincrease or decrease over the course of a particular treatment regime.Changes in dosage may result and become apparent by standard diagnosticassays known in the art. In some instances, chronic administration isrequired. For example, the compounds are administered to the subject inan amount and for duration sufficient to treat the subject.

EXAMPLES

The following non-limiting examples are illustrative of the presentapplication.

Example 1: Exemplary Lipid Vesicle Compositions A. Methods Method 1: InVitro Diffusion Cell Studies:

Full thickness human breast skin was obtained from female donorsundergoing elective mammoplasty surgeries at the Royal UniversityHospital, University of Saskatchewan (Saskatoon, SK, Canada). Approvalfor skin collection was granted by the Human Ethics Committee at theUniversity of Saskatchewan. The skin was collected within 2 h followingsurgery, trimmed of subcutaneous fat, and stored at −20° C. until use.In-line Bronaugh Flow-through diffusion cells with a 9 mm orificediameter (0.63 cm²) were mounted on a water insulated cell warmer(PermeGear, Inc., Hellertown, PA) and set to a constant temperature of32° C. Precut 1 cm² skin sections were placed in the diffusion cellswith the stratum corneum side facing up. Perfusion buffer (100 mMphosphate buffer with 0.05% Na-azide) at 37° C. was circulated throughthe lower half of the diffusion cells at a rate of 1 mL/h using aperistaltic pump. The surface of the skin was dosed with 0.1 mL of theformulations. Following 24 h incubation, the skin samples were removedfrom the cells and their surface was washed 3 times with 10 mL of watereach time. Each skin sample was blot dried and tape-stripped twice usingclear stationary tape to remove surface excess formulation. Skin sampleswere analyzed by UPLC of skin homogenates or by confocal microscopy ofcryosections.

Method 2: Skin Homogenate Preparation for UPLC Analysis

Skin samples were individually homogenized using the gentleMACS™Dissociator (Miltenyi Biotec, Inc., Auburn, Calif.). Each skin sectionwas reconstituted in 1 mL of methanol (for diclofenac samples) or 1 mLacetonitrile (for ibuprofen samples), added to a gentleMACS™ M tube(Miltenyi Biotec, Inc.) and homogenized using the protein extractionprogram (10×55 sec). Samples were then filtered using a 0.2 μm Acrodisc®GH Polypro membrane syringe filter (Pall Corp., Ville St. Laurent, QC,Canada) into 2 mL LC/GC certified clear glass maximum recovery vials(Waters Corp., Milford, Mass.).

The ACQUITY H-class UPLC chromatographic system, consisting of abioQuaternary Solvent Manager, autosampler (bioSample Manager-FlowThrough Needle), variable wavelength UV-detector (photodiode array eλ)and Column Manager, controlled by the Empower 3 software (Waters Corp.)was used for the analysis and method validation for the purpose of thisstudy

Analyses were performed on a 1.7 μm BEH300 C18 50 mm×2.1 mm i.d. column(Waters Corp.) heated to 30° C. (for diclofenac runs) and to 35° C. (foribuprofen runs) with an injection volume of 5 μL. The mobile phase(Solvent A—0.65 methanol: 0.35 milliQ water with pH adjusted to 2.5using phosphoric acid for diclofenac analysis and 0.67 milliQ water:0.34 acetonitrile for ibuprofen analysis) was pumped at 0.45 mL/min (fordiclofenac analysis) and 0.55 mL/min (for ibuprofen analysis), inisocratic mode. The total run time was 5 min and 10 min for diclofenacand ibuprofen analysis, respectively. The mobile phase, standard andsample solutions were filtered through a 0.2 μm Acrodisc® GH Polypromembrane syringe filter (Pall Corp.) and used at room temperature. TheUV detection range was set at 200-260 nm for diclofenac and thecollected data was graphed at 254 nm. For ibuprofen, the UV detectionrange was 200-250 nm and the collected data was graphed at 220 nm. Thecalibration and quantitation (total peak area) were all calculated usingthe Empower 3 software.

Method 3: In Vivo Studies

The animal experiments were approved by the University of WaterlooCommittee on Animal Care Protocol Review Committee. For in vivo deliveryCD1 mice (Charles River) were used. All animals (including controls)were anesthetized with isoflurane and close-shaved a day prior totreatment. The shaved area was cleaned with distilled water usingsterile gauze and dried. Naked plasmid DNA solution or plasmid DNAformulations (50 μL containing 25 μg tD-tomato red fluorescence protein(RFP) coding plasmid for each animal) were applied on the shaved area,and covered with parafilm/Opsite occlusive dressing which was held inplace with a plastic tape for 24 hours. The treated area of the skin wasexcised 24 hours after treatment.

Method 4: Confocal Microscopy

Mouse or human skin samples were characterized using confocal microscopyusing a Zeiss LSM 710 confocal microscope. All samples were embedded inOCT compound matrix and frozen for cryosectioning. Skin samples werecryosectioned with a Leica CM1850 cryostat into 10 μm sections. Confocalmicroscopy images of the skin sections were obtained using a Zeiss LSM710 CLSM using HeNe-laser (543 and 633nm) lines for tdTomato (546/579)and Rhodamine (570/590), 488 nm laser for FITC insulin and FITC-IgG andeither the Plan-Apochromat 20x/0.80 dry objective or the 63x/1.40 oilimmersion objective. Optical zoom selection was applied in selectedcases. Laser intensity, pinhole and gain settings were kept consistentbetween sample sets to enable comparison of relative fluorescenceintensity measurements between different treatments. Images werecaptured and processed using the Zen 2009 software.

The ‘no treatment’ sample was used to confirm gain and pinhole settingsto exclude noise and autofluorescence background for the subsequenttreatment samples.

B: Exemplary Lipid Vesicle Formulation Compositions 1. ExemplaryIbuprofen Lipid Vesicle Formulations

Step 1: Preparation of System A (oil in water emulsion):

System A for exemplary ibuprofen lipid vesicle formulations IB1- IB-6(the oil-in-water sub-micron emulsion) is as follows:

Oil Phase Olive oil   5% Benzalkonium chloride 0.05% Propylparaben 0.05%Glyceryl monostearate NE   1% Cetyl alcohol  0.6% Synchrowax BB4(beeswax) 0.28% Aqueous Phase Ceteth-10   1% Tween 80   1% Methylparaben0.15% Milli-Q Water Qs to 100

Step 2: Procedure preparation of System A (oil-in-water submicronemulsion) preparation (applicable to all formulations):

-   -   1. The oil phase and aqueous phase ingredients were weighed out        in separate beakers.    -   2. Both beakers were heated to ˜70° C. to completely melt and        incorporate all components.    -   3. The water phase was added to the oil phase in one quick        addition, while stirring vigorously with a spatula to form an        o/w crude emulsion, effectively yielding a homogenous milky        solution (˜2min) in the 70° C. water bath.    -   4. The formulation was batch processed using the LV1        Microfluidizer or Nano DeBee homogenizer with Z5 module three        times at 20,000 psi.

Step 3: Preparation of vesicles:

Exemplary Ibuprofen Formulation IB1:

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Oleth-2 1% Ibuprofen 5% Aqueous Phase System A Qs to 100

Procedure for vesicle formation (applicable to all formulations):

-   -   1. The lipid phase components were weighed into a 20mL glass        vial.    -   2. The vial was heated to ˜70° C. in a water bath to completely        melt and incorporate all components.    -   3. The water phase (System A) was added to the liquid phase in        one quick addition.    -   4. The mixture was intermittently vortexed and heated for 5        sec/5 sec for 8-10 cycles until a uniform creamy lotion formed

The following exemplary lipid vesicles formulations were prepared usingthe process described above for Ibuprofen Formulation IB1.

b. Ibuprofen Formulation IB2

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Oleth-2 1% Ibuprofen 5% Aqueous Phase Menthol 1% Camphor 1% System A Qsto 100

Note: Menthol and camphor were premixed without heating, in a glass vialusing a spatula to form a eutectic mixture. After the mixture was fullymixed and in a liquid state, System A was added and vortexed well. Thismixture was then added to the lipid phase as above.

c) Exemplary Ibuprofen Formulation IB3A

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Piperine 0.1%  Oleth-2 1% Ibuprofen 5% Aqueous Phase System A Qs to 100

d) Exemplary Ibuprofen Formulation IB3B

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Piperine 0.1%  Oleth-2 2% Ibuprofen 5% Aqueous Phase System A Qs to 100

e) Exemplary Ibuprofen Formulation IB4A

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Methyl salicylate 2.5%  Oleth-2 1% Ibuprofen 5% Aqueous Phase System AQs to 100

f) Exemplary Ibuprofen Formulation IB4B

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Methyl salicylate 2.5%  Oleth-2 2% Ibuprofen 5% Aqueous Phase System AQs to 100

g) Exemplary Formulation IB5A

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Nerol 1% Oleth-2 1% Ibuprofen 5% Aqueous Phase System A Qs to 100

h) Exemplary Ibuprofen Formulation IB5B

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Nerol 1% Oleth-2 2% Ibuprofen 5% Aqueous Phase System A Qs to 100

i) Exemplary Ibuprofen Formulation IB6A

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Thymol 1% Oleth-2 1% Ibuprofen 5% Aqueous Phase System A Qs to 100

j): Exemplary Ibuprofen Formulation 166B

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Thymol 1% Oleth-2 2% Ibuprofen 5% Aqueous Phase System A Qs to 100

2. Exemplary Diclofenac Lipid Vesicle Formulations

Step 1: Preparation of System A (oil in water sub-micron emulsion)

System A for exemplary diclofenac lipid vesicle formulations DF1 and DF2is as follows:

Oil Phase Olive oil   5% Benzalkonium chloride 0.05% Propyl paraben0.05% Crodamol GMS   1% Cetyl alcohol  0.6% Synchrowax BB4 0.28% AqueousPhase Ceteth-10   1% Tween 80   1% Methyl paraben 0.15% Milli-Q Water Qsto 100

System A was prepared using the process described above for IbuprofenFormulation IB1.

Step 2: Preparation of Vesicles

The following exemplary diclofenac lipid vesicles formulations wereprepared using the process described above for Ibuprofen FormulationIB1.

a) Exemplary Diclofenac Formulation DF1

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Piperine 1% Tween 80 1% PEG-4 dilaurate 1% Diclofenac 5% Aqueous PhaseSystem A Qs to 100

b) Exemplary Diclofenac Formulation DF2

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Methyl Salicylate 2.5%  Tween 80 1% PEG-4 dilaurate 1% Diclofenac 5%Aqueous Phase System A Qs to 1003. Exemplary Peptide and protein lipid vesicle formulations

The following exemplary peptide and protein lipid vesicles formulationswere prepared using the process described above for exemplary ibuprofenformulation IB1.

a) Exemplary 12mer peptide (mwt 1200), insulin (mwt 6000) and IgG(150,000) lipid vesicle formulation 1 (Peptide lipid vesicle formulation1)

Preparation of System A (oil in water sub-micron emulsion) forformulations:

Oil Phase Labrafac CC  5% Glyceryl monostearate NE 1.2% Cetyl alcohol0.6% Synchrowax BB4 (beeswax) 0.3% Propylparaben 0.05%  Aqueous PhaseArlasilk EFA (Phospholipid EFA)  5% Methylparaben 0.15%  Milli-Q WaterQs to 100

Preparation of vesicles:

Lipid Phase Phospholipid (Sunlipon 90H) 7% Cholesterol 1.75%  Monolauroyl lysine 2% Oleth-2 1% Propylene glycol 7% Aqueous PhaseRhodamine-12mer peptide:12mer peptide 0.1%  1:1 OR FITC insulin ORFITC-IgG System A Qs to 100

b) Exemplary 12mer peptide (mwt 1200), insulin (mwt 6000) and IgG(150,000) lipid vesicle formulation 2 (Peptide lipid vesicle formulation2)

Preparation of System A (oil in water sub-micron emulsion)

Oil Phase Labrafac CC  5% Glyceryl monostearate NE 1.2% Cetyl alcohol0.6% Synchrowax BB4 (beeswax) 0.3% Propylparaben 0.05%  Aqueous PhasePolysorbate 80  2% Sorbitan monopalmitate (Span 40) 0.5 Methylparaben0.15%  Milli-Q Water Qs to 100

Preparation of vesicles:

Lipid Phase Phospholipid (Sunlipon 90H) 7% Cholesterol 1.75%   PEG-4dilaurate 1% Propylene glycol 7% Aqueous Phase Rhodamine-12merpeptide:12mer peptide 0.1%  1:1 OR FITC insulin OR FITC-IgG System A Qsto 100

c) Exemplary 12mer peptide (mwt 1200), insulin (mwt 6000) and IgG(150,000) lipid vesicle formulation 3 (Peptide lipid vesicle formulation3)

Preparation of System A (oil in water sub-micron emulsion):

Oil Phase Labrafac CC  5% Glyceryl monostearate NE 1.2% Cetyl alcohol0.6% Synchrowax BB4 (beeswax) 0.3% Propylparaben 0.05%  Aqueous PhasePolysorbate 80  2% Sorbitan monopalmitate (Span 40) 0.5 Methylparaben0.15%  Milli-Q Water Qs to 100

Preparation of vesicles:

Lipid Phase Phospholipid (Sunlipon 90H) 7% Cholesterol 1.75%  Monolauroyl lysine 2% Oleth-2 1% Propylene glycol 7% Aqueous PhaseRhodamine-12mer peptide:12mer peptide 0.1%  1:1 OR FITC insulin ORFITC-IgG System A Qs to 100

d) Examplary 12mer peptide (mwt 1200), insulin (mwt 6000) and IgG(150,000) lipid vesicle formulation 4 (Peptide lipid vesicle formulation4)

Preparation of System A (oil in water sub-micron emulsion) forformulations:

Oil Phase Labrafac CC  5% Glyceryl monostearate NE 1.2% Cetyl alcohol0.6% Synchrowax BB4 (beeswax) 0.3% Propylparaben 0.05%  Aqueous PhaseArlasilk EFA (Phospholipid EFA)  5% Methylparaben 0.15%  Milli-Q WaterQs to 100

Preparation of vesicles:

Lipid Phase Phospholipid (Sunlipon 90H) 7% Cholesterol 1.75%   Oleth-21% Propylene glycol 7% Aqueous Phase Rhodamine-12mer peptide:12merpeptide 0.1%  1:1 OR FITC insulin OR FITC-IgG System A Qs to 100

4) Nucleic Acid Lipid Vesicle Formulations

The following exemplary nucleic acid lipid vesicle lipid vesiclesformulations were prepared using the process described above forexemplary ibuprofen formulation IB1.

a) Comparative plasmid lipid vesicle formulations F-TOM-1

Preparation of System A (oil in water sub-micron emulsion) for F-TOM-1

Oil Phase Olive oil  5% Glyceryl monostearate 1.2% Cetyl alcohol 0.6%Synchrowax BB4 (beeswax) 0.3% Propyl paraben 0.05%  Aqueous PhasePhospholipid EFA  5% Methylparaben 0.1% Milli-Q Water Qs to 100

Preparation of vesicles:

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Aqueous Phase System A Qs to 100

b) Exemplary plasmid formulation lipid vesicle F-TOM-2

Preparation of System A (oil in water sub-micron emulsion) for F-TOM-2

Oil Phase Olive oil  5% Glyceryl monostearate 1.2% Cetyl alcohol 0.6%Synchrowax BB4 (beeswax) 0.3% Propylparaben 0.05%  Aqueous Phase Geminisurfactant 16-3-16 0.1% Tween 80 0.5% Methylparaben 0.1% Milli-Q WaterQs to 100

Preparation of vesicles:

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Aqueous Phase System A Qs to 100

c) Exemplary plasmid lipid vesicle formulation F-TOM-3

Preparation of System A (oil in water sub-micron emulsion) for F-TOM-3

Oil Phase Labrafac CC (medium chain triglycerides) 3% Phospholipid 2%Aqueous Phase Gemini surfactant12-3-12 0.1%  Milli-Q Water Qs to 100

Preparation of vesicles:

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Aqueous Phase System A Qs to 100

d) Exemplary Plasmid lipid vesicle formulation F-TOM-4

Preparation of System A (oil in water sub-micron emulsion) for F-TOM-4

Oil Phase Labrafac CC (medium chain triglycerides) 3% Phospholipid 2%Aqueous Phase Gemini surfactant12-7NCH3-12 0.1%  Milli-Q Water Qs to 100

Preparation of vesicles:

Lipid Phase Phospholipon 90H 10%  Cholesterol 2% Propylene glycol 7%Aqueous Phase System A Qs to 100

e) Exemplary plasmid lipid vesicle formulation F-TOM-5

Preparation of System A (oil in water sub-micron emulsion) for F-TOM-5

Oil Phase Labrafac CC (medium chain triglycerides) 3% Phospholipid 2%Aqueous Phase Gemini surfactant12-7NH-12 0.1%  Milli-Q Water Qs to 100

Preparation of vesicles:

Lipid Phase Phospholipon 90H 10% Lauroyl-capryloyl lysine methyl esther2.5%  Propylene glycol  7% Aqueous Phase System A Qs to 100

C. Results and Discussion Cutaneous Delivery of Ibuprofen and Diclofenac

The results of the in vitro cell diffusion and skin homogenate assays(see Table 3 and Table 4 below) show the improvement of delivery of IBand DF was achieved by incorporating a penetration enhancer componentinto the exemplary biphasic lipid vesicle formulations. It was foundthat adding a hydrophobic non-ionic surfactant with an HLB<10, forexample one with HLB 4-7 such as Oleth-2 enhanced delivery into theviable epidermis. Further enhancement could be achieved when anadditional penetration enhancer such as a terpene (such as menthol,camphor, methylsalicylate) or alkaloid (such as piperine) was added(Table 3). The enhanced permeation effect of the hydrophobic non-ionicsurfactant such as Oleth-2 could be further enhanced by increasing itsconcentration in the formulation (eg. from 1% to 2%) (Table 3).

TABLE 3 Cutaneous Delivery of Ibuprofen. The concentration of IB wasmeasured in the skin homogenates using UPLC. Data presented as average ±s.d. (n = 4). (Whole skin = surface bound drug removed by two D-squamestrips. Stripped skin = viable skin layers only; skin stripped 2 + 10times with D-squame strips) Average amount of IB Average amount of IBSample Formulation type (mg/g skin) (mg/cm² skin IB0* whole skinBiphasic vesicles 0.29 ± 0.018 0.086 ± 0.009  IB0* stripped skin(comparative formula)* 0.30 ± 0.094 0.090 ± 0.05  *same as F-TOM-1 IB1whole skin Biphasic vesicles + Oleth-2 (1%) 0.63 ± 0.108 0.12 ± 0.031IB1 stripped skin 0.54 ± 0.167 0.10 ± 0.049 IB2 whole skin BiphasicvesiclesIB1 + Oleth-2 0.97 ± 0.244 0.17 ± 0.063 IB2 stripped skin (1%) +Menthol + Camphor 0.94 ± 0.266 0.17 ± 0.062 in System A IB3A whole skinBiphasic vesiclesIB1 + 0.928 ± 0.293  0.18 ± 0.078 IB3A stripped skinOleth-2 (1%) + 0.76 ± 0.437 0.15 ± 0.102 piperine in lipid phase IB3Bwhole skin Biphasic vesiclesIB1 + Oleth-2 1.07 ± 0.126 0.20 ± 0.023 IB3Bstripped skin (2%) + piperine 0.72 ± 0.117 0.14 ± 0.043 IB4A whole skinBiphasic vesiclesIB1 + 1.02 ± 0.292 0.19 ± 0.062 IB4A stripped skinOleth-2 (1%) + 1.00 ± 0.385 0.19 ± 0.079 methylsalicylate in lipid phaseIB4B whole skin Biphasic 1.54 ± 0.498 0.27 ± 0.099 IB4B stripped skinvesiclesIB1 + Oleth-2 1.23 ± 0.342 0.22 ± 0.070 (2%) + methylsalicylatein lipid phase IB5A whole skin Biphasic vesiclesIB1 + Oleth-2 0.67 ±0.232 0.17 ± 0.081 IB5A stripped skin (1%) + nerol 0.56 ± 0.322 0.14 ±0.087 in lipid phase IB5B whole skin Biphasic 1.00 ± 0.656 0.19 ± 0.143IB5B stripped skin vesiclesIB1 + Oleth-2 0.71 ± 0.54  0.14 ± 0.118(2%) + nerol in lipid phase IB6A whole skin Biphasic 0.41 ± 0.222 0.08 ±0.055 IB6A stripped skin vesiclesIB1 + Oleth-2 0.42 ± 0.114 0.08 ± 0.035(1%) + thymol in lipid phase IB6B whole skin Biphasic 0.55 ± 0.265 0.12± 0.051 IB6B stripped skin vesiclesIB1 + Oleth-2 0.42 ± 0.131 0.094 ±0.024  (2%) + thymol in lipid phase

TABLE 4 Cutaneous Delivery of Diclofenac: The concentration of DF wasmeasured in the skin homogenates using UPLC. Data presented as average ±s.d. (n = 4). (Whole skin = surface bound drug removed by two D-squamestrips. Stripped skin = viable skin layers only; skin stripped 2 + 10times with D-squame strips) Average amount if DF Average amount of DFSample Formulation type (mg/g skin) (mg/cm²) DF1 whole skin Biphasic0.72 ± 0.544 0.20 ± 0.191 DF1 stripped skin vesicle + Piperine + Tween0.51 ± 0.372 0.13 ± 0.010 80 + PEG-4 dilaurate in lipid phase DF2 wholeskin Biphasic 0.39 ± 0.258 0.11 ± 0.083 DF2 stripped skin vesicle +methylsalicylate + Tween  0.72 ± 0.802* 0.21 ± 0.247 80 + PEG-4dilaurate in lipid phase *this data was relatively variable

Cutaneous Delivery of Peptide and Protein Therapeutic Agents

The cryosections of human skin samples treated in vitro in diffusioncells with topical formulations containing fluorescence labelledpeptides and proteins were evaluated for the presence of fluorescentprotein. The enhancement of delivery of protein and peptide compounds isshown with three compounds of increasing molecular weight (FIG. 1 ). Itwas shown that the incorporation of a penetration enhancer hydrophobicnon-ionic surfactant with HLB<10 (eg. Oleth-2, sorbitan monopalmitate[Span 40], or PEG-4 dilaurate) increased the delivery of these proteinsand peptides (FIG. 1 ). Table 5 indicates the relative fluorescenceintensity of measured in the viable epidermal layers. While all of thesehydrophobic non-ionic surfactants with HLB<10 were effective in deliveryenhancement in the biphasic vesicles, the enhancement level was asfollows (from highest to lowest): PEFA/Oleth-2>Tween 80/Span40Oleth-2 >Tween 80/Span 40/PEG-4-dilaurate>PEFA/PEG-4-dilaurate. (Thesurfactant in italics is present in oil and water emulsion component ofthe comparative biphasic vesicles for the emulsification function; thesurfactant in bold indicates the additional penetration enhancer for thepenetration enhancer function).

TABLE 5 Average relative fluorescence intensity values obtained from theconfocal microscopic images Average relative fluorescence intensity inFormulation the viable epidermal layer F1- TAMRA-13mer peptide (mwt1440) 24 F2- TAMRA-13mer peptide (mwt 1440) 40 F3- TAMRA-13mer peptide(mwt 1200) 50 F4- TAMRA-13mer peptide (mwt 1440) 212 F1-FITC-insulin(mwt 6,000) 10 F2-FITC-insulin (mwt 6,000) 40 F3-FITC-insulin (mwt6,000) 15 F4-FITC-insulin (mwt 6,000) 20 F1-FITC-IgG (mwt 150,000) 15F2-FITC-IgG (mwt 150,000) 20 F3-FITC-IgG (mwt 150,000) 40 F4-FITC-IgG(mwt 150,000) 50

Cutaneous Delivery of Nucleic Acids

Mouse skin samples treated with topical formulations containing plasmidDNA encoding the red tdTomato reporter gene were evaluated for theexpression of tdTomato red fluorescent protein. Compared to thecomparative biphasic vesicle (F-TOM-1 containing monocationic surfactantPEFA) the other formulations containing a replacement of PEFA, ie.dicationic gemini surfactants as complexing agents for the negativelycharged plasmid DNA increased the delivery of plasmid DNA and thecutaneous gene expression in vivo in mice. All dicationic geminisurfactants used were effective in the delivery of plasmid DNA whenincorporated into the biphasic vesicle structure. Enhancement was asfollows (from highest to lowest): F-TOM-5 dicationic gemini surfactant12-7NH-12/phospholipid emulsifier>F-TOM-4 dicationic gemini surfactant12-7CH3-12/phospholipid emulsifier>F-TOM-3 dicationic gemini surfactant12-3-12/phospholipid emulsifier>F-TOM-2* Tween 80/dicationic geminisurfactant 16-3-16 (surfactant in italics is an improved functionalsurfactant for biphasic vesicles to improve the encapsulation of highlynegatively charged nucleic acids; surfactant in bold indicates the addedHLB <10 synergistic penetration enhancer function) (Table 6). *F-TOM-2is a variation for control formulation where the original biphasicvesicles prepared with Tween 80/PEFA were modified to Tween 80/geminisurfactant.

All blank samples showed little to none background fluorescence (FIG. 2). Samples treated with intradermal naked pDNA show a significant amountof tdTomato expression (images not shown). For each formulation threepanels are shown: the first panel: red channel for RFP expression (seenas light colored areas in the epidermis and dermis); second panel:general tissue stain (blue nuclear stain Syto 60); third panel: mergedimage).

TABLE 6 Average relative fluorescence intensity values obtained from theconfocal microscopic images Average relative fluorescence intensity inthe viable epidermal layer Formulation (range) F-TOM-1 (comparative20-30 biphasic vesicles) F-TOM-2  50-100 F-TOM-3 30-50 F-TOM-4  50-100F-TOM-5 200-250

The present disclosure is not to be limited in terms of the particularembodiments described in this application. Many modifications andvariations can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. Functionally equivalentmethods and compositions within the scope of the disclosure, in additionto those enumerated herein, will be apparent to those skilled in the artfrom the foregoing descriptions. Such modifications and variations areintended to fall within the scope of the appended claims. The presentdisclosure is to be limited only by the terms of the appended claims,along with the full scope of equivalents to which such claims areentitled. It is to be understood that this disclosure is not limited toparticular methods, reagents, compounds compositions or biologicalsystems, which can of course vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

All publications, patent applications, issued patents, and otherdocuments referred to in this specification are herein incorporated byreference as if each individual publication, patent application, issuedpatent, or other document was specifically and individually indicated tobe incorporated by reference in its entirety. Definitions that arecontained in text incorporated by reference are excluded to the extentthat they contradict definitions in this disclosure.

While certain embodiments have been illustrated and described, it shouldbe understood that changes and modifications can be made therein inaccordance with ordinary skill in the art without departing from thetechnology in its broader aspects as defined in the claims providedbelow.

1. A method of generating biphasic lipid vesicles, comprising: a.preparing an oil-in-water emulsion by mixing one or more oil componentsof the oil-in-water emulsion with one or more aqueous components of theoil-in-water emulsion; b. solubilizing one or more vesicle forminglipids in a solvent other than water; c. adding one or more penetrationenhancing agents to the oil components, the aqueous components, or thesolubilized vesicle forming lipids, wherein the one or more penetrationenhancing agents comprise one or more non-ionic surfactants having ahydrophilic-lipophilic balance (HLB) of about 10 or less; d. adding theoil-in-water emulsion to the solubilized vesicle forming lipids; and e.mixing the oil-in-water emulsion and the solubilized vesicle forminglipids under mixing conditions effective to form the biphasic lipidvesicles comprising a lipid bilayer, the oil-in-water emulsion entrappedin the biphasic lipid vesicles, and the one or more penetrationenhancing agents entrapped in the oil-in-water emulsion or the lipidbilayer.
 2. The method of claim 1, wherein the one or more penetrationenhancing agents are entrapped in the oil-in-water emulsion.
 3. Themethod of claim 1, wherein the one or more penetration enhancing agentsare entrapped in the lipid bilayer and the oil-in-water emulsion.
 4. Themethod of claim 1, wherein the lipid bilayer comprises about 10 wt %,about 9 wt %, about 8 wt %, about7 wt %, about 6 wt %, about 5 wt %,about 4 wt %, about3 wt %, about 2 wt %, about 1 wt %, about 0.5 wt % orabout0.1 wt % of the one or more penetration enhancing agents.
 5. Themethod of claim 1, wherein the one or more non-ionic surfactants havinga hydrophilic-lipophilic balance (HLB) of about 10 or less comprisespolyethylene glycol ethers of fatty alcohols, sorbitan esters,polysorbates, sorbitan esters, polyethylene glycol fatty acid esters, orany combination thereof.
 6. The method of claim 5, wherein thepolyethylene glycol ethers of fatty alcohols having the HLB of about 10or less comprise diethylene glycol hexadecyl ether,2-(2-octadecoxyethoxy)ethanol, diethylene glycol monooleyl ether,polyoxyethylene (3) oleyl ether, polyoxyethylene (5) oleyl ether, or anycombination thereof.
 7. The method of claim 5, wherein the polyethyleneglycol fatty acid esters having the HLB of about 10 or less comprisePEG-8 dilaurate, PEG-4 dilaurate, PEG-4 laurate, PEG-8 dioleate, PEG-8distearate, PEG-8 distearate, PEG-7 glyceryl cocoate, PEG-20 almondglycerides, or any combination thereof.
 8. The method of claim 1,wherein the one or more non-ionic surfactants having the H LB of about10 or less comprise an oleyl ether having the HLB of about 10 or less.9. The method of claim 1, wherein the one or more penetration enhancingagents further comprise one or more terpenes, one or more alkaloids, oneor more salicylate derivatives, one or more polycationic surfactants, orany combination thereof.
 10. The method of claim 9, wherein the terpenescomprise eugenol, d-limonene, menthol, menthone, farnesol, neridol,camphor, nerol, thymol, or any combination thereof.
 11. The method ofclaim 9, wherein the salicylate derivatives comprise ethyl salicylate,salicylic acid, acetylsalicylic acid, trolamine salicylate, or anycombination thereof.
 12. The method of claim 9, wherein the alkaloidscomprise piperine, lobeline, caffeine, theobromine theophylline,nicotine, colchicine, N-methyl pyrrolidone, hygrine, capsaicin,berberine, sanguinarine, histamine, pilocarpine, or any combinationthereof.
 13. The method of claim 9, wherein the polycationic surfactantscomprise one or more gemini cationic surfactants and the one or moregemini cationic surfactants are of a quaternary ammonium type.
 14. Themethod of claim 9, wherein the polycationic surfactants comprisepolycationic amino acids.
 15. The method of claim 1, wherein the one ormore penetration enhancing agents having the HLB of about 10 or lesscomprise diethylene glycol monooleyl ether, PEG-4 dilaurate, sorbitanmonopalmitate, or any combination thereof.
 16. The method of claim 1,wherein the oil-in-water emulsion of the biphasic lipid vesicles isstabilized by one or more surfactant comprising polyoxyethylene (10)cetyl ether and polysorbate
 80. 17. The method of claim 1, thesolubilized vesicle forming lipids comprise one or more ofphospholipids, glycolipids, lecithins, or ceramides.
 18. The method ofclaim 1, further comprising adding one or more compounds in (b).
 19. Themethod of claim 18, wherein the one or more compounds are entrapped inoil-in-water emulsion of the biphasic lipid vesicle, lipid bilayer ofthe biphasic lipid vesicle or both the oil-in-water emulsion and thelipid bilayer.
 20. The method of claim 18, wherein the one or morecompounds comprise a small molecule, protein, peptide, carbohydrate,nucleic acid, vaccine antigen, plant extract, or any combinationthereof.